Targeting pax2 for the induction of defbi-mediated tumor immunity and cancer therapy

ABSTRACT

Provided is a method of treating cancer in a subject by inhibiting expression of PAX2. An example of a cancer treated by the present method is prostate cancer. In the cancer treatment methods disclosed, the method of inhibiting expression of PAX2 can be by administration of a nucleic acid encoding an siRNA for PAX2. A method of treating cancer in a subject by administering DEFB1 is also provided. Similarly, provided is a method of treating cancer in a subject by increasing expression of DEFB1 in the subject.

The present application claims priority to U.S. Patent Application No.60/726,921, filed Oct. 14, 2005, which application is incorporatedherein fully by this reference.

This invention was made with United States Government support under NIHNCI Grant Number: CA096788-02. The Government may have certain rights inthe invention.

BRIEF DESCRIPTION OF INVENTION

Current anticancer chemotherapies that are based on alkylating agents,anti-metabolites and natural products are heterogeneous in theirmechanism of action. Consequently, most of them also act against normalcells resulting in severe side effects and toxicity to the patient.Disclosed herein is a method for the treatment of advanced prostatecancer using human beta defensin-1 (DEFB1), which is a naturallycomponent of the innate immune system, to induce prostate cancer tumorimmunity. This is accomplished through endogenously added DEFB1,ectopically expressed DEFB1 or de novo expression of EFB1 by inhibitingthe transcriptional repressor PAX2 by a variety of mechanisms or agents.Inhibiting PAX2 expression by siRNA therapy turns on DEFB1 expressionand generates DEFB1-mediated cell death in prostate cancer. With this,the technology described here is used for the design of small moleculesto specifically block PAX2 expression. Alternatively provided aremolecules containing the CCTTG (SEQ ID NO:1) recognition sequence (ineither forward of reverse orientation) that to bind to the DNA-bindingdomain of PAX2 preventing its binding to the DEFB1 promoter throughcompetitive inhibition. This permits DEFB1 expression, triggering bothan innate and adaptive immune response, and resulting in the killing ofprostate cancer cells and the suppression of prostate tumor formation.In conclusion, these modulators of innate tumor immunity, PAX2 andDEFB1, and the molecular therapies based on them provide for thetreatment of prostate cancer with little toxicity to the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate one or more embodiments of theinvention and, together with the written description, serve to explainthe principles of the invention. Wherever possible, the same referencenumbers are used throughout the drawings to refer to the same or likeelements of an embodiment.

FIG. 1. QRT-PCR analysis of DEFB1 Expression. In order to verifyinduction of DEFB1 expression, QRT-PCR was performed. A, DEFB1 relativeexpression levels were compared in clinical samples from 6 patients thatunderwent radical prostatectomies. B, DEFB1 relative expression levelswere compared in benign and malignant prostatic clinical samples, hPrECcells and in prostate cancer cell lines before and after DEFB1induction. C, DEFB1 relative expression levels were analyzed in benigntissue, malignant tissue and PIN in a single tissue section. D, DEFB1expression in benign tissue, malignant tissue and PIN in one patient wascompared to the average DEFB1 expression level found in benign tissue.

FIG. 2. Microscopic analysis of DEFB1 induced changes in membraneintegrity and cell morphology. Cell morphology of DU145, PC3 and LNCaPwas analyzed by phase contrast microscopy after 48 hours of DEFB1induction. Membrane ruffling is indicated by black arrows and apoptoticbodies are indicated white arrows.

FIG. 3. Analysis of DEFB1 Cytotoxicity in Prostate Cancer Cells. Theprostate cell lines DU145, PC3 and LNCaP were treated with PonA toinduce DEFB1 expression for 1-3 days after which MTT assay was performedto determine cell viability. Results represent mean±s.d., n=9.

FIG. 4. Induction of cell death in DU145 and PC3 cells by DEFB1. DEFB1expression was induced in prostate cancer cell lines DU145 (A) and PC3(B) and then subjected to annexin V/FITC/propidium iodide staining andflow cytometric analysis. Cells positive for propidium iodide andannexin V were considered apoptotic. Times of induction are shown undereach panel. Numbers next to the boxes for each time point represent thepercentages of propidium iodide (PI)⁻ annexin V⁺ cells (lower rightquadrant), and PI⁺ annexin V⁺ cells (upper right quadrant). The data arefrom a single experiment that is representative of three separateexperiments.

FIG. 5. Pan-caspase analysis following DEFB1 induction. DU145 and PC3cells were stained with FAM-VAD-FMK-labeled fluoromethyl ketone todetect caspase activity. Cells were visible under DIC for eachcondition. Confocal microscopic analysis revealed no caspase staining incontrol DU145 (B), PC3 cells (F) and LNCaP (J). Cells treated with PonAfor 24 hours to induce DEFB1 revealed caspase activity in DU145 (D) andPC3 (H). No caspase activity was detected in LNCaP (L).

FIG. 6. Silencing of PAX2 Protein Expression Following PAX2 siRNATreatment. (a) Western blot analysis of PC3 and DU145 cells transfectedwith PAX2 siRNA duplex at day zero (lane 1), day two (lane 2), and dayfour (lane 3). (b) Western blot analysis of PC3 and DU145 cellstransfected with PAX2 siRNA duplex at day zero (lane 1), day two (lane2), day four (lane 3) and day 6 (lane 4). PAX2 protein was undetectableas early as after four days of treatment (lane 3) in DU145 cells andafter six days of treatment in PC3. Blots were stripped and re-probedfor β-actin as an internal control.

FIG. 7. Analysis of Prostate Cancer Cells Growth after Treatment withPax 2 siRNA. Phase contrast microscopic analysis of DU145, PC3 and LNCaPat 6 days in the presence of normal growth media. Treatment withnegative control siRNA had no effect on the cells. However, there was asignificant reduction in cell number in all three lines followingtreatment with PAX2 siRNA.

FIG. 8. Analysis of Cell Death Following siRNA Silencing of PAX2.Prostate cancer cell lines PC3, DU145, and LNCaP were treated with 0.5μg of a pool of four PAX2 siRNA's or four non-specific control siRNA'sfor 2, 4 or 6 days after which MTT assay was done to determine cellviability. Results represent mean±s.d., n=9.

FIG. 9. Analysis of Caspase Activity. DU145, PC3 and LNCaP cells werestained with carboxyfluorescein-labeled fluoromethyl ketone to detectedcaspase activity following treatment with PAX2 siRNA. Confocalmicroscopic analysis of untreated and treated cells show cells werevisible with DIC. Analysis under fluorescence revealed no caspasestaining in control DU145 (B), PC3 cells (F) and LNCaP cells (J).However, cell treated with PAX2 siRNA induced caspase activity in DU145(D), PC3 (H) and LNCaP (L).

FIG. 10. Analysis of Apoptotic Factors Following PAX2 siRNA Treatment.Changes in expression of pro-apoptotic factors were compared inuntreated control cells and in cells treated for six days with PAX2siRNA. A, BAX expression levels increased in DU145, PC3 and LNCaP. B,BID expression increased in DU145 and LNCaP, but change in PC3. C, BADexpression levels increased in all three cell lines.

FIG. 11. Model of PAX2 Binding to DNA Recognition Sequence. The PAX2transcriptional repressor binds to a CCTTG (SEQ ID NO:1) recognitionsite immediately adjacent to the DEFB1 TATA box preventing transcriptionand DEFB1 protein expression. Inhibition of PAX2 protein expressionallows normal DEFB1 expression.

FIG. 12. Illustration of the DEFB1 Reporter Construct. The DEFB1promoter consisting of the first 160 bases upstream of the mRNA startsite was PCR amplified from DU145 cell and ligated into the pGL3luciferase reporter plasmid.

FIG. 13. Inhibition of PAX2 Results in DEFB1 Expression. DU145, PC3,LNCaP and HPrEC were treated for 48 hours with PAX2 siRNA. QRT-PCRanalysis before treatment showed no DEFB1 expression in DU145, PC3 andLNCaP. However, DEFB1 expression was restored following treatment in alllines. There was no change in DEFB1 expression following siRNA treatmentof PAX2-null HprEC.

FIG. 14. Inhibition of PAX2 Results in Increased DEFB1 PromoterActivity. PC3 promoter/pGL3 and DU145 promoter/pGL3 construct weregenerated and were transfected into PC3 and DU145 cells, respectively.Promoter activity was compared before and after PAX2 inhibition by siRNAtreatment. DEFB1 promoter activity increased 2.65-fold in DU145 and 3.78fold in PC3 following treatment.

FIG. 15. DEFB1 Causes Loss of Membrane Integrity. Membrane integrity ofPC3 and DU145 cells was analyzed by confocal laser microscopy followingthe induction of DEFB1 expression for 48 hours. Green staining wasindicative of the localization of AO, and red staining represents EtBr.Yellow staining represents the co-localization of both AO and EtBr inthe nucleus.

FIG. 16. PAX2 Inhibition Results in Loss of Membrane Integrity. Cellswere treated for 48 hours with PAX2 siRNA and membrane integrity wasanalyzed by confocal laser microscopy.

FIG. 17. ChIP Analysis of PAX2 binding to DEFB1 Promoter. ChIP analysiswas performed on DU145 and PC3 cells. Following immunoprecipitation withan anti-PAX2 antibody, PCR was performed to detect the DEFB1 promoterregion containing the GTTCC (SEQ ID NO: 2) PAX2 recognition site. Thisdemonstrates that the PAX2 transcriptional repressor is bound to theDEFB1 promoter in prostate cancer cell lines.

FIG. 18. Predicted Structure of the PrdPD and PrdHD with DNA. Thecoordinates of the structures of the PrdPD bound to DNA (Xu et al., 1995and the PrdHD bound to DNA (Wilson et al., 1995) were used to constructa model of the two domains as they bound to a PH0 site. The individualbinding sites are abutted next to each other with a specific orientationas indicated. The PAI binding site is in red, the HD binding site is inblue, and the corresponding PAI domain is in turquoise, and HD is inyellow. The RED domain is oriented based on the PrdPD crystal structure.

FIG. 19. Comparison of Consensus Sequences of Different Paired Domains.At the top of the Figure is drawn a schematic representation ofprotein±DNA contacts described in the crystallographic analysis of thePrd-paired-domain±DNA complex [9]. Empty boxes indicate a-helices,shaded boxes indicates b-sheets and a thick line indicate a b-turn.Contacting amino acids are shown by single-letter code. Only directamino acid±base contacts are shown. Empty circles indicate major groovecontacts while red arrows indicate minor groove contacts. This scheme isaligned to all known consensus sequences for paired-domain proteins (topstrands only are shown). Vertical lines between consensus sequencesindicate conserved base-pairs. Numbering of the positions is shown atthe bottom of the Figure and it is the same as that used in [9].

DETAILED DESCRIPTION OF THE INVENTION

As shown herein, PAX2 inhibits expression of DEFB1, and DEFB1 is shownto have tumor cell killing activity. Thus, provided is a method oftreating cancer in a subject by inhibiting expression of PAX2. Anexample of a cancer treated by the present method is prostate cancer.The present methods are particularly effective for treatment of latestage prostate cancer.

In the cancer treatment methods disclosed, the method of inhibitingexpression of PAX 2 can be by administration of a nucleic acid encodinga siRNA for PAX 2. Dharmachon is a commercial source for such siRNAs.

The siRNA for use in the methods can be selected from the groupconsisting of:

AUAGACUCGACUUGACUUCUU (SEQ ID NO: 3) AUCUUCAUCACGUUUCCUCUU (SEQ ID NO:4) GUAUUCAGCAAUCUUGUCCUU (SEQ ID NO: 5) GAUUUGAUGUGCUCUGAUGUU (SEQ IDNO: 6)

The following table illustrates the above antisense sequences and theircorresponding sense sequences.

Sense (5′-3′) AntiSense (5′-3′) Sequence A 5′-GAAGUCAAGUCGAGUCUAUUU-3′5′-AUAGACUCGACUUGACUUCUU-3′ (SEQ ID NO: 7) (SEQ ID NO: 3) Sequence B5′-GAGGAAACGUGAUGAAGAUUU-3′ 5′-AUCUUCAUCACGUUUCCUCUU-3′ (SEQ ID NO: 8)(SEQ ID NO: 4) Sequence C 5′-GGACAAGAUUGCUGAAUACUU-3′5′-GUAUUCAGCAAUCUUGUCCUU-3′ (SEQ ID NO: 9) (SEQ ID NO: 5) Sequence D5′-CAUCAGAGCA-CAUCAAAUCUU-3′ 5′-GAUUUGAUGUGCUCUGAUGUU-3′ (SEQ ID NO: 10)(SEQ ID NO: 6)

Further examples of molecules that inhibit PAX2 include: #1ACCCGACTATGTTCGCCTGG (SEQ ID NO: 11), #2 AAGCTCTGGATCGAGTCTTTG (SEQ IDNO: 12), and #4 ATGTGTCAGGCACACAGACG (SEQ ID NO: 13). #4 was shown toinhibit PAX2 (Davies et al., Hum. Mol. Gen January 15, 13 (2); 235).

Another paper (Muratovska et al., Paired-Box genes are frequentlyexpressed in cancer and often required for cancer cell survival Oncogene(2003) 22, 7989-7997) discloses the following siRNAs:GUCGAGUCUAUCUGCAUCCUU (SEQ ID NO: 14) and GGAUGCAGAUAGACUCGACUU (SEQ IDNO: 15).

To down-regulate Pax2 expression, Fonsato et al. transfectedtumor-derived endothelial cells with an anti-sense PAX2 vector. SeeFonsato V. et al (Expression of Pax2 in human renal tumor-derivedendothelial cells sustains apoptosis resistance and angiogenesis, Am JPathol. 2006 February; 168(2):706-1), incorporated herein by referenefor its description of this molecule. Similarly, Hueber et al. teachthat PAX2 antisense cDNA and PAX2-small interfering RNA (100 nM) reduceendogenous PAX2 protein. See Hueber et al. PAX2 inactivation enhancescisplatin-induced apoptosis in renal carcinoma cells, Kidney Int. 2006April; 69(7):1139-45 incorporated herein for its teaching of PAX2antisense and PAX2 siRNA.

Additional inhibitors of PAX2 expression or the binding of PAX2 to theDEFB1 promoter are provided to increase DEFB1 expression in thepresently disclosed methods. For example, small molecules and antibodiesare designed based on the present studies to interfere with or inhibitthe binding of PAX2 to the DEFB1 promoter.

As shown herein, PAX2 inhibits expression of DEFB1, and DEFB1 is shownto have tumor cell killing activity. Thus, a method of treating cancerin a subject by administering DEFB1 is also provided. An example of acancer treated by the present method is prostate cancer.

Similarly, provided is a method of treating cancer in a subject byincreasing expression of DEFB1 in the subject. The present methods ofadministering or increasing the expression of DEFB1 are particularlyeffective for treatment of late stage prostate cancer.

In one embodiment of the methods of the invention for treating cancer byadministering DEFB1 or increasing DEFB1 expression (e.g., by inhibitingexpression or binding of PAX2), the subject is a subject diagnosed withprostate cancer. In a further embodiment of the methods of the inventionfor treating cancer by administering DEFB1 or increasing DEFB1expression (e.g., by inhibiting expression or binding of PAX2), thesubject is a subject diagnosed with advanced (late stage) prostatecancer.

In the method wherein the expression of DEFB1 is increased, it can beincreased by blocking the binding of PAX2 to the DEFB1 promoter. Theblocking of binding of PAX2 to the DEFB1 promoter can be byadministration of an oligonucleotide containing the PAX2 DNA bindingsite of DEFB1. This oligonucleotide can be complementary to the sequenceof PAX2 that binds to the DEFB1 promoter. Alternatively, theoligonucleotide can interact with the PAX2 in a way that inhibitsbinding to DEFB1. This interaction can be based on three-dimensionalstructure rather than primary nucleotide sequence.

PAX proteins are a family of transcription factors conserved duringevolution and able to bind specific DNA sequences through a domainscalled a “paired domain” and a “homeodomain”. The paired domain (PD) isa consensus sequence shared by certain PAX proteins (e.g., PAX2 andPAX6). The PD directs DNA binding of amino acids located in the α3-helixforming a DNA-Protein complex. For PAX2, the amino acids in the HDrecognize and interact specifically with a CCTTG (SEQ ID NO: 1) DNA coresequence. Therefore, the critical region for PAX2 binding to DEFB1 wouldbe AAGTTCACCCTTGACTGTG (SEQ ID NO: 16). Oligonucleotides up to andexceeding 64 bases in length, which include this sequence or itscomplement are expected to be inhibitors.

The DNA-binding specificity of the PAX-8 paired domain was investigated.Site selection experiments indicate that PAX-8 binds to a consensussequence similar to those bound by PAX-2 and PAX-5. When consensussequences of various paired domains are observed in light of recentstructural studies describing paired-domain-DNA interaction [Xu, Rould,Jun, Desplan and Pabo (1995) Cell 80, 639-650], it appears thatbase-pairs contacted in the minor groove are conserved, while most ofthe base-pairs contacted in the major groove are not. Therefore anetwork of specific minor groove contacts is a common characteristic ofpaired-domain-DNA interactions. The functional importance of such anetwork can be successfully tested by analyzing the effect ofconsensus-based mutations on the PAX2 binding site of the DEFB1promoter.

The PAX2 DNA binding site of DEFB1 can comprise SEQ ID NO:1 (CCTTG).

The oligonucleotide comprising to the PAX2 DNA binding site of DEFB1 isselected from the group consisting of

X1 CCTTG X₂ (SEQ ID NO: 17), wherein X₁=NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN and is/are from 1 to 35 contiguous flankingnucleotides of DEFB1 and X₂=NNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNN andis/are from 1 to 35 contiguous flanking nucleotides of DEFB1. Thenucleotides can be contiguous nucleotides that normally flank the PAX2DNA binding site of DEFB1. Alternatively, they can be unrelated toDEFB1, and selected routinely to avoid interference with the recognitionsequence.

For example, the inhibitory oligonucleotides can be selected from thegroup consisting of:

(SEQ ID NO: 18) CTCCCTTCAGTTCCGTCGAC (SEQ ID NO: 19)CTCCCTTCACCTTGGTCGAC (SEQ ID NO: 20)ACTGTGGCACCTCCCTTCAGTTCCGTCGACGAGGTTGTGC (SEQ ID NO: 21)ACTGTGGCACCTCCCTTCACCTTGGTCGACGAGGTTGTGC

The disclosed compositions can be used to treat any disease whereuncontrolled cellular proliferation occurs such as cancers. Anon-limiting list of different types of cancers is as follows: lymphomas(Hodgkins and non-Hodgkins), leukemias, carcinomas, carcinomas of solidtissues, squamous cell carcinomas, adenocarcinomas, sarcomas, gliomas,high grade gliomas, blastomas, neuroblastomas, plasmacytomas,histiocytomas, melanomas, adenomas, hypoxic tumors, myelomas,AIDS-related lymphomas or sarcomas, metastatic cancers, or cancers ingeneral.

A representative but non-limiting list of cancers that the disclosedcompositions can be used to treat is the following: lymphoma, B celllymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloidleukemia, bladder cancer, brain cancer, nervous system cancer, head andneck cancer, squamous cell carcinoma of head and neck, kidney cancer,lung cancers such as small cell lung cancer and non-small cell lungcancer, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer,prostate cancer, skin cancer, liver cancer, melanoma, squamous cellcarcinomas of the mouth, throat, larynx, and lung, colon cancer,cervical cancer, cervical carcinoma, breast cancer, and epithelialcancer, renal cancer, genitourinary cancer, pulmonary cancer, esophagealcarcinoma, head and neck carcinoma, large bowel cancer, hematopoieticcancers; testicular cancer; colon and rectal cancers, prostatic cancer,or pancreatic cancer. Compounds disclosed herein may also be used forthe treatment of precancer conditions such as cervical and analdysplasias, other dysplasias, severe dysplasias, hyperplasias, atypicalhyperplasias, and neoplasias. Further, a number of diseases stemmingfrom chronic inflammation, e.g., prostatitis and Benign ProstaticHypertrophy (BPH), as well as various cancers of the prostate, can beimpacted by the present methods and compounds.

DEFB1's gene locus (8p23.3) is a hotspot for deletions and has beenlinked to patients with poorer prognosis. Thus, DEFB1 (and perhaps PAX2)can be used as a biomarker, e.g., in a screening for the early detectionof prostate cancer. Furthermore, data presented here indicate that itsloss may occur as early as PIN (or even before), and may be a majorcontributing factor to the onset of prostate cancer.

Nucleic Acid Homology/Identity/Similarity

It is understood that as discussed herein the use of the terms homologyand identity mean the same thing as similarity. Thus, for example, ifthe use of the word homology is used between two non-natural sequencesit is understood that this is not necessarily indicating an evolutionaryrelationship between these two sequences, but rather is looking at thesimilarity or relatedness between their nucleic acid sequences. Many ofthe methods for determining homology between two evolutionarily relatedmolecules are routinely applied to any two or more nucleic acids orproteins for the purpose of measuring sequence similarity regardless ofwhether they are evolutionarily related or not.

In general, it is understood that one way to define any known variantsand derivatives or those that might arise, of the disclosed genes andproteins herein, is through defining the variants and derivatives interms of homology to specific known sequences. This identity ofparticular sequences disclosed herein is also discussed elsewhereherein. In general, variants of genes and proteins herein disclosedtypically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, or 99 percent homology to the stated sequence or the nativesequence. Those of skill in the art readily understand how to determinethe homology of two proteins or nucleic acids, such as genes. Forexample, the homology can be calculated after aligning the two sequencesso that the homology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment. It isunderstood that any of the methods typically can be used and that incertain instances the results of these various methods may differ, butthe skilled artisan understands if identity is found with at least oneof these methods, the sequences would be said to have the statedidentity, and be disclosed herein.

For example, as used herein, a sequence recited as having a particularpercent homology to another sequence refers to sequences that have therecited homology as calculated by any one or more of the calculationmethods described above. For example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingthe Zuker calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by any of theother calculation methods. As another example, a first sequence has 80percent homology, as defined herein, to a second sequence if the firstsequence is calculated to have 80 percent homology to the secondsequence using both the Zuker calculation method and the Pearson andLipman calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by the Smith andWaterman calculation method, the Needleman and Wunsch calculationmethod, the Jaeger calculation methods, or any of the other calculationmethods. As yet another example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingeach of calculation methods (although, in practice, the differentcalculation methods will often result in different calculated homologypercentages).

Hybridization/Selective hybridization

The term hybridization typically means a sequence driven interactionbetween at least two nucleic acid molecules, such as an oligonucleotideinhibitor, a primer or a probe and a gene. Sequence driven interactionmeans an interaction that occurs between two nucleotides or nucleotideanalogs or nucleotide derivatives in a nucleotide specific manner. Forexample, G interacting with C or A interacting with T are sequencedriven interactions. Typically sequence driven interactions occur on theWatson-Crick face or Hoogsteen face of the nucleotide. The hybridizationof two nucleic acids is affected by a number of conditions andparameters known to those of skill in the art. For example, the saltconcentrations, pH, and temperature of the reaction all affect whethertwo nucleic acid molecules will hybridize.

Parameters for selective hybridization between two nucleic acidmolecules are well known to those of skill in the art. For example, insome embodiments selective hybridization conditions can be defined asstringent hybridization conditions. For example, stringency ofhybridization is controlled by both temperature and salt concentrationof either or both of the hybridization and washing steps. For example,the conditions of hybridization to achieve selective hybridization mayinvolve hybridization in high ionic strength solution (6×SSC or 6×SSPE)at a temperature that is about 12-25° C. below the Tm (the meltingtemperature at which half of the molecules dissociate from theirhybridization partners) followed by washing at a combination oftemperature and salt concentration chosen so that the washingtemperature is about 5° C. to 20° C. below the Tm. The temperature andsalt conditions are readily determined empirically in preliminaryexperiments in which samples of reference DNA immobilized on filters arehybridized to a labeled nucleic acid of interest and then washed underconditions of different stringencies. Hybridization temperatures aretypically higher for DNA-RNA and RNA-RNA hybridizations. The conditionscan be used as described above to achieve stringency, or as is known inthe art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989;Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is hereinincorporated by reference for material at least related to hybridizationof nucleic acids). A preferable stringent hybridization condition for aDNA:DNA hybridization can be at about 68° C. (in aqueous solution) in6×SSC or 6×SSPE followed by washing at 68° C. Stringency ofhybridization and washing, if desired, can be reduced accordingly as thedegree of complementarity desired is decreased, and further, dependingupon the G-C or A-T richness of any area wherein variability is searchedfor. Likewise, stringency of hybridization and washing, if desired, canbe increased accordingly as homology desired is increased, and further,depending upon the G-C or A-T richness of any area wherein high homologyis desired, all as known in the art.

Another way to define selective hybridization is by looking at theamount (percentage) of one of the nucleic acids bound to the othernucleic acid. For example, in some embodiments selective hybridizationconditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid isbound to the non-limiting nucleic acid. Typically, the non-limitingnucleic acid is in for example, 10 or 100 or 1000 fold excess. This typeof assay can be performed at under conditions where both the limitingand non-limiting nucleic acid are for example, 10 fold or 100 fold or1000 fold below their k_(d), or where only one of the nucleic acidmolecules is 10 fold or 100 fold or 1000 fold or where one or bothnucleic acid molecules are above their k_(d).

Another way to define selective hybridization is by looking at thepercentage of nucleic acid that gets enzymatically manipulated underconditions where hybridization is required to promote the desiredenzymatic manipulation, e.g., for primers. For example, in someembodiments selective hybridization conditions would be when at leastabout, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100percent of the primer is enzymatically manipulated under conditionswhich promote the enzymatic manipulation, for example if the enzymaticmanipulation is DNA extension, then selective hybridization conditionswould be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100 percent of the primer molecules are extended. Preferredconditions also include those suggested by the manufacturer or indicatedin the art as being appropriate for the enzyme performing themanipulation.

Just as with homology, it is understood that there are a variety ofmethods herein disclosed for determining the level of hybridizationbetween two nucleic acid molecules. It is understood that these methodsand conditions may provide different percentages of hybridizationbetween two nucleic acid molecules, but unless otherwise indicatedmeeting the parameters of any of the methods would be sufficient. Forexample if 80% hybridization was required and as long as hybridizationoccurs within the required parameters in any one of these methods it isconsidered disclosed herein.

It is understood that those of skill in the art understand that if acomposition or method meets any one of these criteria for determininghybridization either collectively or singly it is a composition ormethod that is disclosed herein.

Nucleotides and Related Molecules

A nucleotide is a molecule that contains a base moiety, a sugar moietyand a phosphate moiety. Nucleotides can be linked together through theirphosphate moieties and sugar moieties creating an internucleosidelinkage. The base moiety of a nucleotide can be adenin-9-yl (A),cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T).The sugar moiety of a nucleotide is a ribose or a deoxyribose. Thephosphate moiety of a nucleotide is pentavalent phosphate. Anon-limiting example of a nucleotide would be 3′-AMP (3′-adenosinemonophosphate) or 5′-GMP (5′-guanosine monophosphate).

A nucleotide analog is a nucleotide which contains some type ofmodification to any of the base, sugar, or phosphate moieties.Modifications to the base moiety would include natural and syntheticmodifications of A, C, G, and T/U as well as different purine orpyrimidine bases, such as uracil-5-yl (.psi.), hypoxanthin-9-yl (I), and2-aminoadenin-9-yl. A modified base includes but is not limited to5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional basemodifications can be found for example in U.S. Pat. No. 3,687,808,Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and Sanghvi, Y. S., Chapter 15, Antisense Research andApplications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRCPress, 1993. Certain nucleotide analogs, such as 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine can increase the stability of duplex formation. Oftentime base modifications can be combined with for example a sugarmodification, such as 2′-O-methoxyethyl, to achieve unique propertiessuch as increased duplex stability. There are numerous United Statespatents such as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, which detail and describe a range of basemodifications. Each of these patents is herein incorporated byreference.

Nucleotide analogs can also include modifications of the sugar moiety.Modifications to the sugar moiety would include natural modifications ofthe ribose and deoxyribose as well as synthetic modifications. Sugarmodifications include but are not limited to the following modificationsat the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-,S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl andalkynyl may be substituted or unsubstituted C₁ to C₁₀, alkyl or C₂ toC₁₀ alkenyl and alkynyl. 2′ sugar modifications also include but are notlimited to —O[(CH₂)_(n)O]_(m)CH₃, —O(CH₂)_(n)OCH₃, —O(CH₂)_(n)NH₂,—O(CH₂)_(n)CH₃, —O(CH₂)_(n)—ONH₂, and —O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂,where n and m are from 1 to about 10.

Other modifications at the 2′ position include but are not limited to:C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃,SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. Similar modifications mayalso be made at other positions on the sugar, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide. Modifiedsugars would also include those that contain modifications at thebridging ring oxygen, such as CH₂ and S. Nucleotide sugar analogs mayalso have sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. There are numerous United States patents thatteach the preparation of such modified sugar structures such as U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference in its entirety.

Nucleotide analogs can also be modified at the phosphate moiety.Modified phosphate moieties include but are not limited to those thatcan be modified so that the linkage between two nucleotides contains aphosphorothioate, chiral phosphorothioate, phosphorodithioate,phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates including 3′-alkylene phosphonate and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates. It is understood that these phosphate or modifiedphosphate linkage between two nucleotides can be through a 3′-5′ linkageor a 2′-5′ linkage, and the linkage can contain inverted polarity suchas 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and freeacid forms are also included. Numerous United States patents teach howto make and use nucleotides containing modified phosphates and includebut are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is hereinincorporated by reference.

It is understood that nucleotide analogs need only contain a singlemodification, but may also contain multiple modifications within one ofthe moieties or between different moieties.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize nucleic acids in a Watson-Crick or Hoogsteen manner,but which are linked together through a moiety other than a phosphatemoiety. Nucleotide substitutes are able to conform to a double helixtype structure when interacting with the appropriate target nucleicacid.

Nucleotide substitutes are nucleotides or nucleotide analogs that havehad the phosphate moiety and/or sugar moieties replaced. Nucleotidesubstitutes do not contain a standard phosphorus atom. Substitutes forthe phosphate can be for example, short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts. Numerous United States patents disclosehow to make and use these types of phosphate replacements and includebut are not limited to U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,56.1,225;5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439,each of which is herein incorporated by reference.

It is also understood in a nucleotide substitute that both the sugar andthe phosphate moieties of the nucleotide can be replaced, by for examplean amide type linkage (aminoethylglycine) (PNA). U.S. Pat. Nos.5,539,082; 5,714,331;and 5,719,262 teach how to make and use PNAmolecules, each of which is herein incorporated by reference. (See alsoNielsen et al., Science, 1991, 254, 1497-1500).

It is also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance for example, cellularuptake. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Such conjugates include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al.,Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937. Numerous United States patents teach thepreparation of such conjugates and include, but are not limited to U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,each of which is herein incorporated by reference.

A Watson-Crick interaction is at least one interaction with theWatson-Crick face of a nucleotide, nucleotide analog, or nucleotidesubstitute. The Watson-Crick face of a nucleotide, nucleotide analog, ornucleotide substitute includes the C2, N1, and C6 positions of a purinebased nucleotide, nucleotide analog, or nucleotide substitute and theC2, N3, C4 positions of a pyrimidine based nucleotide, nucleotideanalog, or nucleotide substitute.

A Hoogsteen interaction is the interaction that takes place on theHoogsteen face of a nucleotide or nucleotide analog, which is exposed inthe major groove of duplex DNA. The Hoogsteen face includes the N7position and reactive groups (NH2 or O) at the C6 position of purinenucleotides.

Sequences

There are a variety of sequences related to the DEFB1 gene and to thePAX2 transcriptional factor, respectively, having the following GenBankAccession Numbers: U50930 and NM_(—)003989.1. These sequences and othersare herein incorporated by reference in their entireties as well as forindividual subsequences contained therein.

The one particular sequence set forth in SEQ ID NO: 64 and havingGenBank accession number U50930 is used herein as an example toexemplify a source for the disclosed DEFB1 nucleic acids. The oneparticular sequence set forth in SEQ ID NO: 46 and having GenBankaccession number NM_(—)003989.1 is used herein as an example, toexemplify a source for the disclosed PAX2 nucleic acids. Other examplesof PAX2 sequences, based on alternative splicing are also found inGenBank. These are variants a-e, shown in Appendices B-F. It isunderstood that the description related to this sequence is applicableto any sequence related to unless specifically indicated otherwise.Those of skill in the art understand how to resolve sequencediscrepancies and differences and to adjust the compositions and methodsrelating to a particular sequence to other related sequences. siRNAmolecules, competitive inhibitors of DEFB1 promoter-PAX2, and primersand/or probes can be designed for any DEFB1 or PAX2 sequence given theinformation disclosed herein and known in the art.

Nucleic Acid Synthesis

The nucleic acids, such as, the oligonucleotides to be used asinhibitors can be made using standard chemical synthesis methods or canbe produced using enzymatic methods or any other known method. Suchmethods can range from standard enzymatic digestion followed bynucleotide fragment isolation (see for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) topurely synthetic methods, for example, by the cyanoethyl phosphoramiditemethod using a Milligen or Beckman System 1Plus DNA synthesizer (forexample, Model 8700 automated synthesizer of Milligen-Biosearch,Burlington, Mass. or ABI Model 380B). Synthetic methods useful formaking oligonucleotides are also described by Ikuta et al., Ann. Rev.Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triestermethods), and Narang et al., Methods Enzymol., 65:610-620 (1980),(phosphotriester method). Protein nucleic acid molecules can be madeusing known methods such as those described by Nielsen et al.,Bioconjug. Chem. 5:3-7 (1994).

Primers and Probes

Disclosed are compositions including primers and probes, which arecapable of interacting with the DEFB1 gene as disclosed herein. Incertain embodiments the primers are used to support DNA amplificationreactions. Typically the primers will be capable of being extended in asequence specific manner. Extension of a primer in a sequence specificmanner includes any methods wherein the sequence and/or composition ofthe nucleic acid molecule to which the primer is hybridized or otherwiseassociated directs or influences the composition or sequence of theproduct produced by the extension of the primer. Extension of the primerin a sequence specific manner therefore includes, but is not limited to,PCR, DNA sequencing, DNA extension, DNA polymerization, RNAtranscription, or reverse transcription. Techniques and conditions thatamplify the primer in a sequence specific manner are preferred. Incertain embodiments the primers are used for the DNA amplificationreactions, such as PCR or direct sequencing. It is understood that incertain embodiments the primers can also be extended using non-enzymatictechniques, where for example, the nucleotides or oligonucleotides usedto extend the primer are modified such that they will chemically reactto extend the primer in a sequence specific manner.

The size of the primers or probes for interaction with the DEFB1 or PAX2gene in certain embodiments can be any size that supports the desiredenzymatic manipulation of the primer, such as DNA amplification or thesimple hybridization of the probe or primer. A typical primer or probewould be at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275,300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750,3000, 3500, or 4000 nucleotides long.

In other embodiments a primer or probe can be less than or equal to 6,7, 8, 9, 10, 11, 12 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000nucleotides long.

The primers for the DEFB1 or PAX2 gene typically will be used to producean amplified DNA product that contains the region of the DEFB1 gene towhich PAX2 binds. In general, typically the size of the product will besuch that the size can be accurately determined to within 3, or 2 or 1nucleotides.

In certain embodiments this product is at least 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000nucleotides long.

In other embodiments the product is less than or equal to 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or4000 nucleotides long.

Functional Nucleic Acids

RNAi

It is also understood that the disclosed nucleic acids can be used forRNAi or RNA interference. It is thought that RNAi involves a two-stepmechanism for RNA interference (RNAi): an initiation step and aneffector step. For example, in the first step, input double-stranded(ds) RNA (siRNA) is processed into small fragments, such as21-23-nucleotide ‘guide sequences’. RNA amplification occurs in wholeanimals. Typically then, the guide RNAs can be incorporated into aprotein RNA complex which is capable of degrading RNA, the nucleasecomplex, which has been called the RNA-induced silencing complex (RISC).This RISC complex acts in the second effector step to destroy mRNAs thatare recognized by the guide RNAs through base-pairing interactions. RNAiinvolves the introduction by any means of double stranded RNA into thecell which triggers events that cause the degradation of a target RNA.RNAi is a form of post-transcriptional gene silencing. In addition tothe siRNAs disclosed herein, disclosed are RNA hairpins that can act inRNAi. For description of making and using RNAi molecules see, e.g.,Hammond et al., Nature Rev Gen 2: 110-119 (2001); Sharp, Genes Dev 15:485-490 (2001), Waterhouse et al., Proc. Natl. Acad. Sci. USA 95(23):13959-13964 (1998) all of which are incorporated herein by reference intheir entireties and at least form material related to delivery andmaking of RNAi molecules.

RNAi has been shown to work in many types of cells, including mammaliancells. For work in mammalian cells it is preferred that the RNAmolecules which will be used as targeting sequences within the RISCcomplex are shorter. For example, less than or equal to 50 or 40 or 30or 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, or 10 nucleotides in length. These RNA molecules can also haveoverhangs on the 3′ or 5′ ends relative to the target RNA which is to becleaved. These overhangs can be at least or less than or equal to 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 nucleotides long. RNAi works inmammalian stem cells, such as mouse ES cells. Examples of siRNAs can befound in Table 4.

Functional nucleic acids are nucleic acid molecules that have a specificfunction, such as binding a target molecule or catalyzing a specificreaction. Functional nucleic acid molecules can be divided into thefollowing categories, which are not meant to be limiting. For example,functional nucleic acids include antisense molecules, aptamers,ribozymes, triplex forming molecules, and external guide sequences. Thefunctional nucleic acid molecules can act as effectors, inhibitors,modulators, and stimulators of a specific activity possessed by a targetmolecule, or the functional nucleic acid molecules can possess a de novoactivity independent of any other molecules.

Functional nucleic acid molecules can interact with any macromolecule,such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functionalnucleic acids can interact with mRNA or the genomic DNA of PAX2. Oftenfunctional nucleic acids are designed to interact with other nucleicacids based on sequence homology between the target molecule and thefunctional nucleic acid molecule. In other situations, the specificrecognition between the functional nucleic acid molecule and the targetmolecule is not based on sequence homology between the functionalnucleic acid molecule and the target molecule, but rather is based onthe formation of tertiary structure that allows specific recognition totake place.

Antisense molecules are designed to interact with a target nucleic acidmolecule through either canonical or non-canonical base pairing. Theinteraction of the antisense molecule and the target molecule isdesigned to promote the destruction of the target molecule through, forexample, RNAseH mediated RNA-DNA hybrid degradation. Alternatively theantisense molecule is designed to interrupt a processing function thatnormally would take place on the target molecule, such as transcriptionor replication. Antisense molecules can be designed based on thesequence of the target molecule. Numerous methods for optimization ofantisense efficiency by finding the most accessible regions of thetarget molecule exist. Exemplary methods would be in vitro selectionexperiments and DNA modification studies using DMS and DEPC. It ispreferred that antisense molecules bind the target molecule with adissociation constant (k_(d)) less than or equal to 10⁻⁶, 10⁻⁸, 10⁻¹⁰,or 10⁻¹². A representative sample of methods and techniques which aid inthe design and use of antisense molecules can be found in the followingnon-limiting list of U.S. Pat. Nos. 5,135,917, 5,294,533, 5,627,158,5,641,754, 5,691,317, 5,780,607, 5,786,138, 5,849,903, 5,856,103,5,919,772, 5,955,590, 5,990,088, 5,994,320, 5,998,602, 6,005,095,6,007,995, 6,013,522, 6,017,898, 6,018,042, 6,025,198, 6,033,910,6,040,296, 6,046,004, 6,046,319, and 6,057,437.

Aptamers are molecules that interact with a target molecule, preferablyin a specific way. Typically aptamers are small nucleic acids rangingfrom 15-50 bases in length that fold into defined secondary and tertiarystructures, such as stem-loops or G-quartets. Aptamers can bind smallmolecules, such as ATP (U.S. Pat. No. 5,631,146) and theophiline (U.S.Pat. No. 5,580,737), as well as large molecules, such as reversetranscriptase (U.S. Pat. No. 5,786,462) and thrombin (U.S. Pat. No.5,543,293). Aptamers can bind very tightly with k_(d)s from the targetmolecule of less than 10⁻¹² M. It is preferred that the aptamers bindthe target molecule with a k_(d) less than 10⁻⁶, 10⁻⁸, 10⁻¹⁰, or 10⁻¹².Aptamers can bind the target molecule with a very high degree ofspecificity. For example, aptamers have been isolated that have greaterthan a 10000 fold difference in binding affinities between the targetmolecule and another molecule that differ at only a single position onthe molecule (U.S. Pat. No. 5,543,293). It is preferred that the aptamerhave a k_(d) with the target molecule at least 10, 100, 1000, 10,000, or100,000 fold lower than the k_(d) with a background binding molecule.Representative examples of how to make and use aptamers to bind avariety of different target molecules can be found in the followingnon-limiting list of U.S. Pat. Nos. 5,476,766, 5,503,978, 5,631,146,5,731,424, 5,780,228, 5,792,613, 5,795,721, 5,846,713, 5,858,660,5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,011,020,6,013,443, 6,020,130, 6,028,186, 6,030,776, and 6,051,698.

Ribozymes are nucleic acid molecules that are capable of catalyzing achemical reaction, either intramolecularly or intermolecularly.Ribozymes are thus catalytic nucleic acid. It is preferred that theribozymes catalyze intermolecular reactions. There are a number ofdifferent types of ribozymes that catalyze nuclease or nucleic acidpolymerase type reactions which are based on ribozymes found in naturalsystems, such as hammerhead ribozymes, (for example, but not limited tothe following U.S. Pat. Nos. 5,334,711, 5,436,330, 5,616,466, 5,633,133,5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288,5,891,683, 5,891,684, 5,985,621, 5,989,908, 5,998,193, 5,998,203, WO9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO9718312 by Ludwig and Sproat) hairpin ribozymes (for example, but notlimited to the following U.S. Pat. Nos. 5,631,115, 5,646,031, 5,683,902,5,712,384, 5,856,188, 5,866,701, 5,869,339, and 6,022,962), andtetrahymena ribozymes (for example, but not limited to the followingU.S. Pat. Nos. 5,595,873 and 5,652,107). There are also a number ofribozymes that are not found in natural systems, but which have beenengineered to catalyze specific reactions de novo (for example, but notlimited to the following U.S. Pat. Nos. 5,580,967, 5,688,670, 5,807,718,and 5,910,408). Preferred ribozymes cleave RNA or DNA substrates, andmore preferably cleave RNA substrates. Ribozymes typically cleavenucleic acid substrates through recognition and binding of the targetsubstrate with subsequent cleavage. This recognition is often basedmostly on canonical or non-canonical base pair interactions. Thisproperty makes ribozymes particularly good candidates for targetspecific cleavage of nucleic acids because recognition of the targetsubstrate is based on the target substrates sequence. Representativeexamples of how to make and use ribozymes to catalyze a variety ofdifferent reactions can be found in the following non-limiting list ofU.S. Pat. Nos. 5,646,042, 5,693,535, 5,731,295, 5,811,300, 5,837,855,5,869,253, 5,877,021, 5,877,022, 5,972,699, 5,972,704, 5,989,906, and6,017,756.

Triplex forming functional nucleic acid molecules are molecules that caninteract with either double-stranded or single-stranded nucleic acid.When triplex molecules interact with a target region, a structure calleda triplex is formed, in which there three strands of DNA are forming acomplex dependant on both Watson-Crick and Hoogsteen base-pairing.Triplex molecules are preferred because they can bind target regionswith high affinity and specificity. It is preferred that the triplexforming molecules bind the target molecule with a k_(d) less than 10⁻⁶,10⁻⁸, 10⁻¹⁰, or 10⁻¹². Representative examples of how to make and usetriplex forming molecules to bind a variety of different targetmolecules can be found in the following non-limiting list of U.S. Pat.Nos. 5,176,996, 5,645,985, 5,650,316, 5,683,874, 5,693,773, 5,834,185,5,869,246, 5,874,566, and 5,962,426.

External guide sequences (EGSs) are molecules that bind a target nucleicacid molecule forming a complex, and this complex is recognized by RNaseP, which cleaves the target molecule. EGSs can be designed tospecifically target a RNA molecule of choice. RNAse P aids in processingtransfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited tocleave virtually any RNA sequence by using an EGS that causes the targetRNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 byYale, and Forster and Altman, Science 238:407-409 (1990)).

Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can beutilized to cleave desired targets within eukaryotic cells. (Yuan etal., Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); WO 93/22434 byYale; WO 95/24489 by Yale; Yuan and Altman, EMBO J 14:159-168 (1995),and Carrara et al., Proc. Natl. Acad. Sci. (USA) 92:2627-2631 (1995)).Representative examples of how to make and use EGS molecules tofacilitate cleavage of a variety of different target molecules be foundin the following non-limiting list of U.S. Pat. Nos. 5,168,053,5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162.

Delivery of the Compositions to Cells

There are a number of compositions and methods which can be used todeliver nucleic acids to cells, either in vitro or in vivo. Thesemethods and compositions can largely be broken down into two classes:viral based delivery systems and non-viral based delivery systems. Forexample, the nucleic acids can be delivered through a number of directdelivery systems such as, electroporation, lipofection, calciumphosphate precipitation, plasmids, viral vectors, viral nucleic acids,phage nucleic acids, phages, cosmids, or via transfer of geneticmaterial in cells or carriers such as cationic liposomes. Appropriatemeans for transfection, including viral vectors, chemical transfectants,or physico-mechanical methods such as electroporation and directdiffusion of DNA, are described by, for example, Wolff, J. A., et al.,Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818,(1991)Such methods are well known in the art and readily adaptable foruse with the compositions and methods described herein. In certaincases, the methods will be modified to specifically function with largeDNA molecules. Further, these methods can be used to target certaindiseases and cell populations by using the targeting characteristics ofthe carrier.

Nucleic Acid Based Delivery Systems

Transfer vectors can be any nucleotide construction used to delivergenes into cells (e.g., a plasmid), or as part of a general strategy todeliver genes, e.g., as part of recombinant retrovirus or adenovirus(Ram et al. Cancer Res. 53:83-88, (1993)).

As used herein, plasmid or viral vectors are agents that transport thedisclosed nucleic acids, such as DEFB1 coding sequences, PAX2 siRNAs orother antisense molecules into the cell without degradation and includea promoter yielding expression of the gene in the cells into which it isdelivered. Viral vectors are, for example, Adenovirus, Adeno-associatedvirus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronaltrophic virus, Sindbis and other RNA viruses, including these viruseswith the HIV backbone. Also preferred are any viral families which sharethe properties of these viruses which make them suitable for use asvectors. Retroviruses include Murine Maloney Leukemia virus, MMLV, andretroviruses that express the desirable properties of MMLV as a vector.Retroviral vectors are able to carry a larger genetic payload, i.e., atransgene or marker gene, than other viral vectors, and for this reasonare a commonly used vector. However, they are not as useful innon-proliferating cells. Adenovirus vectors are relatively stable andeasy to work with, have high titers, and can be delivered in aerosolformulation, and can transfect non-dividing cells. Pox viral vectors arelarge and have several sites for inserting genes, they are thermostableand can be stored at room temperature. A preferred embodiment is a viralvector which has been engineered so as to suppress the immune responseof the host organism, elicited by the viral antigens. Preferred vectorsof this type will carry coding regions for Interleukin 8 or 10.

Viral vectors can have higher transaction (ability to introduce genes)abilities than chemical or physical methods to introduce genes intocells. Typically, viral vectors contain, nonstructural early genes,structural late genes, an RNA polymerase III transcript, invertedterminal repeats necessary for replication and encapsidation, andpromoters to control the transcription and replication of the viralgenome. When engineered as vectors, viruses typically have one or moreof the early genes removed and a gene or gene/promotor cassette isinserted into the viral genome in place of the removed viral DNA.Constructs of this type can carry up to about 8 kb of foreign geneticmaterial. The necessary functions of the removed early genes aretypically supplied by cell lines which have been engineered to expressthe gene products of the early genes in trans.

Retroviral Vectors

A retrovirus is an animal virus belonging to the virus family ofRetroviridae, including any types, subfamilies, genus, or tropisms.Retroviral vectors, in general, are described by Verma, I. M.,Retroviral vectors for gene transfer. In Microbiology-1985, AmericanSociety for Microbiology, pp. 229-232, Washington, (1985), which isincorporated by reference herein. Examples of methods for usingretroviral vectors for gene therapy are described in U.S. Pat. Nos.4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136;and Mulligan, (Science 260:926-932 (1993)); the teachings of which areincorporated herein by reference.

A retrovirus is essentially a package which has packed into it nucleicacid cargo. The nucleic acid cargo carries with it a packaging signal,which ensures that the replicated daughter molecules will be efficientlypackaged within the package coat. In addition to the package signal,there are a number of molecules which are needed in cis, for thereplication, and packaging of the replicated virus. Typically aretroviral genome, contains the gag, pol, and env genes which areinvolved in the making of the protein coat. It is the gag, pol, and envgenes which are typically replaced by the foreign DNA that it is to betransferred to the target cell. Retrovirus vectors typically contain apackaging signal for incorporation into the package coat, a sequencewhich signals the start of the gag transcription unit, elementsnecessary for reverse transcription, including a primer binding site tobind the tRNA primer of reverse transcription, terminal repeat sequencesthat guide the switch of RNA strands during DNA synthesis, a purine richsequence 5′ to the 3′ LTR that serve as the priming site for thesynthesis of the second strand of DNA synthesis, and specific sequencesnear the ends of the LTRs that enable the insertion of the DNA state ofthe retrovirus to insert into the host genome. The removal of the gag,pol, and env genes allows for about 8 kb of foreign sequence to beinserted into the viral genome, become reverse transcribed, and uponreplication be packaged into a new retroviral particle. This amount ofnucleic acid is sufficient for the delivery of a one to many genesdepending on the size of each transcript. It is preferable to includeeither positive or negative selectable markers along with other genes inthe insert.

Since the replication machinery and packaging proteins in mostretroviral vectors have been removed (gag, pol, and env), the vectorsare typically generated by placing them into a packaging cell line. Apackaging cell line is a cell line which has been transfected ortransformed with a retrovirus that contains the replication andpackaging machinery, but lacks any packaging signal. When the vectorcarrying the DNA of choice is transfected into these cell lines, thevector containing the gene of interest is replicated and packaged intonew retroviral particles, by the machinery provided in cis by the helpercell. The genomes for the machinery are not packaged because they lackthe necessary signals.

Adenoviral Vectors

The construction of replication-defective adenoviruses has beendescribed (Berkner et al., J. Virology 61:1213-1220 (1987); Massie etal., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987);Zhang “Generation and identification of recombinant adenovirus byliposome-mediated transfection and PCR analysis” BioTechniques15:868-872 (1993)). The benefit of the use of these viruses as vectorsis that they are limited in the extent to which they can spread to othercell types, since they can replicate within an initial infected cell,but are unable to form new infectious viral particles. Recombinantadenoviruses have been shown to achieve high efficiency gene transferafter direct, in vivo delivery to airway epithelium, hepatocytes,vascular endothelium, CNS parenchyma and a number of other tissue sites(Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin.Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092(1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992);Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout,Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993);Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen.Virology 74:501-507 (1993)). Recombinant adenoviruses achieve genetransduction by binding to specific cell surface receptors, after whichthe virus is internalized by receptor-mediated endocytosis, in the samemanner as wild type or replication-defective adenovirus (Chardonnet andDales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985);Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell.Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991);Wickham et al., Cell 73:309-319 (1993)).

A viral vector can be one based on an adenovirus which has had the E1gene removed and these virions are generated in a cell line such as thehuman 293 cell line. In another preferred embodiment both the E1 and E3genes are removed from the adenovirus genome.

Adeno-Associated Viral Vectors

Another type of viral vector is based on an adeno-associated virus(AAV). This defective parvovirus is a preferred vector because it caninfect many cell types and is nonpathogenic to humans. AAV type vectorscan transport about 4 to 5 kb and wild type AAV is known to stablyinsert into chromosome 19. Vectors which contain this site specificintegration property are preferred. An especially preferred embodimentof this type of vector is the P4.1 C vector produced by Avigen, SanFrancisco, Calif., which can contain the herpes simplex virus thymidinekinase gene, HSV-tk, and/or a marker gene, such as the gene encoding thegreen fluorescent protein, GFP.

In another type of AAV virus, the AAV contains a pair of invertedterminal repeats (ITRs) which flank at least one cassette containing apromoter which directs cell-specific expression operably linked to aheterologous gene. Heterologous in this context refers to any nucleotidesequence or gene which is not native to the AAV or B19 parvovirus.

Typically the AAV and B19 coding regions have been deleted, resulting ina safe, noncytotoxic vector. The AAV ITRs, or modifications thereof,confer infectivity and site-specific integration, but not cytotoxicity,and the promoter directs cell-specific expression. U.S. Pat. No.6,261,834 is herein incorporated by reference for material related tothe AAV vector.

1. The disclosed vectors thus provide DNA molecules which are capable ofintegration into a mammalian chromosome without substantial toxicity.

2. The inserted genes in viral and retroviral usually contain promoters,and/or enhancers to help control the expression of the desired geneproduct. A promoter is generally a sequence or sequences of DNA thatfunction when in a relatively fixed location in regard to thetranscription start site. A promoter contains core elements required forbasic interaction of RNA polymerase and transcription factors, and maycontain upstream elements and response elements.

Large Payload Viral Vectors

Molecular genetic experiments with large human herpes viruses haveprovided a means whereby large heterologous DNA fragments can be cloned,propagated and established in cells permissive for infection with herpesviruses (Sun et al., Nature genetics 8: 33-41, 1994; Cotter andRobertson, Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses(herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have thepotential to deliver fragments of human heterologous DNA>150 kb tospecific cells. EBV recombinants can maintain large pieces of DNA in theinfected B-cells as episomal DNA. Individual clones carried humangenomic inserts up to 330 kb appeared genetically stable. Themaintenance of these episomes requires a specific EBV nuclear protein,EBNA1, constitutively expressed during infection with EBV. Additionally,these vectors can be used for transfection, where large amounts ofprotein can be generated transiently in vitro. Herpesvirus ampliconsystems are also being used to package pieces of DNA>220 kb and toinfect cells that can stably maintain DNA as episomes.

Other useful systems include, for example, replicating andhost-restricted non-replicating vaccinia virus vectors.

Non-Nucleic Acid Based Systems

The disclosed compositions can be delivered to the target cells in avariety of ways. For example, the compositions can be delivered throughelectroporation, or through lipofection, or through calcium phosphateprecipitation. The delivery mechanism chosen will depend in part on thetype of cell targeted and whether the delivery is occurring for examplein vivo or in vitro.

Thus, the compositions can comprise, in addition to the disclosedvectors for example, lipids such as liposomes, such as cationicliposomes (e.g., DOTMA, DOPE, and DC-cholesterol) or anionic liposomes.Liposomes can further comprise proteins to facilitate targeting aparticular cell, if desired. Administration of a composition comprisinga compound and a cationic liposome can be administered to the bloodafferent to a target organ or inhaled into the respiratory tract totarget cells of the respiratory tract. Regarding liposomes, see, e.g.,Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Feigner etal. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat.No.4,897,355. Furthermore, the compound can be administered as acomponent of a microcapsule that can be targeted to specific cell types,such as macrophages, or where the diffusion of the compound or deliveryof the compound from the microcapsule is designed for a specific rate ordosage.

In the methods described above which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), delivery of the compositions to cells canbe via a variety of mechanisms. As one example, delivery can be via aliposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the disclosednucleic acid or vector can be delivered in vivo by electroporation, thetechnology for which is available from Genetronics, Inc. (San Diego,Calif.) as well as by means of a SONOPORATION machine (ImaRxPharmaceutical Corp., Tucson, Ariz.).

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). These techniques can be used for avariety of other specific cell types. Vehicles such as “stealth” andother antibody conjugated liposomes (including lipid mediated drugtargeting to colonic carcinoma), receptor mediated targeting of DNAthrough cell specific ligands, lymphocyte directed tumor targeting, andhighly specific therapeutic retroviral targeting of murine glioma cellsin vivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellular, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

Nucleic acids that are delivered to cells which are to be integratedinto the host cell genome, typically contain integration sequences.These sequences are often viral related sequences, particularly whenviral based systems are used. These viral integration systems can alsobe incorporated into nucleic acids which are to be delivered using anon-nucleic acid based system of deliver, such as a liposome, so thatthe nucleic acid contained in the delivery system can be come integratedinto the host genome.

Other general techniques for integration into the host genome include,for example, systems designed to promote homologous recombination withthe host genome. These systems typically rely on sequence flanking thenucleic acid to be expressed that has enough homology with a targetsequence within the host cell genome that recombination between thevector nucleic acid and the target nucleic acid takes place, causing thedelivered nucleic acid to be integrated into the host genome. Thesesystems and the methods necessary to promote homologous recombinationare known to those of skill in the art.

In vivo/ex vivo

As described herein, the compositions can be administered in apharmaceutically acceptable carrier and can be delivered to thesubject's cells in vivo and/or ex vivo by a variety of mechanisms wellknown in the art (e.g., uptake of naked DNA, liposome fusion,intramuscular injection of DNA via a gene gun, endocytosis and thelike).

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The compositions can be introduced into the cells via anygene transfer mechanism, such as, for example, calcium phosphatemediated gene delivery, electroporation, microinjection orproteoliposomes. The transduced cells can then be infused (e.g., in apharmaceutically acceptable carrier) or homotopically transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

Expression Systems

The nucleic acids that are delivered to cells typically containexpression controlling systems. For example, the inserted genes in viraland retroviral systems usually contain promoters, and/or enhancers tohelp control the expression of the desired gene product. A promoter isgenerally a sequence or sequences of DNA that function when in arelatively fixed location in regard to the transcription start site. Apromoter contains core elements required for basic interaction of RNApolymerase and transcription factors, and may contain upstream elementsand response elements.

Viral Promoters and Enhancers

Preferred promoters controlling transcription from vectors in mammalianhost cells may be obtained from various sources, for example, thegenomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. beta actin promoter. Theearly and late promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment which also contains the SV40 viral originof replication (Fiers et al., Nature, 273: 113 (1978)). The immediateearly promoter of the human cytomegalovirus is conveniently obtained asa HindIII E restriction fragment (Greenway, P. J. et al., Gene 18:355-360 (1982)). Of course, promoters from the host cell or relatedspecies also are useful herein.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3′(Lusky, M. L., et al., Mol. Cell Bio. 3: 1108 (1983)) to thetranscription unit. Furthermore, enhancers can be within an intron(Banerji, J. L. et al., Cell 33: 729 (1983)) as well as within thecoding sequence itself (Osborne, T. F., et al., Mol. Cell Bio. 4: 1293(1984)). They are usually between 10 and 300 by in length, and theyfunction in cis. Enhancers function to increase transcription fromnearby promoters. Enhancers also often contain response elements thatmediate the regulation of transcription. Promoters can also containresponse elements that mediate the regulation of transcription.Enhancers often determine the regulation of expression of a gene. Whilemany enhancer sequences are now known from mammalian genes (globin,elastase, albumin, -fetoprotein and insulin), typically one will use anenhancer from a eukaryotic cell virus for general expression. Preferredexamples are the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

The promotor and/or enhancer may be specifically activated either bylight or specific chemical events which trigger their function. Systemscan be regulated by reagents such as tetracycline and dexamethasone.There are also ways to enhance viral vector gene expression by exposureto irradiation, such as gamma irradiation, or alkylating chemotherapydrugs.

In certain embodiments the promoter and/or enhancer region can act as aconstitutive promoter and/or enhancer to maximize expression of theregion of the transcription unit to be transcribed. In certainconstructs the promoter and/or enhancer region be active in alleukaryotic cell types, even if it is only expressed in a particular typeof cell at a particular time. A preferred promoter of this type is theCMV promoter (650 bases). Other preferred promoters are SV40 promoters,cytomegalovirus (full length promoter), and retroviral vector LTR.

It has been shown that all specific regulatory elements can be clonedand used to construct expression vectors that are selectively expressedin specific cell types such as melanoma cells. The glial fibrillaryacetic protein (GFAP) promoter has been used to selectively expressgenes in cells of glial origin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contain a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases. It is alsopreferred that the transcribed units contain other standard sequencesalone or in combination with the above sequences improve expressionfrom, or stability of, the construct.

Proteins

Protein Variants

Variants of the DEFB1 protein are provided. Derivatives of the DEFB1protein function in the disclosed methods and compositions. Proteinvariants and derivatives are well understood to those of skill in theart and in can involve amino acid sequence modifications. For example,amino acid sequence modifications typically fall into one or more ofthree classes: substitutional, insertional or deletional variants.Insertions include amino and/or carboxyl terminal fusions as well asintrasequence insertions of single or multiple amino acid residues.Insertions ordinarily will be smaller insertions than those of amino orcarboxyl terminal fusions, for example, on the order of one to fourresidues. Immunogenic fusion protein derivatives, such as thosedescribed in the examples, are made by fusing a polypeptide sufficientlylarge to confer immunogenicity to the target sequence by cross-linkingin vitro or by recombinant cell culture transformed with DNA encodingthe fusion. Deletions are characterized by the removal of one or moreamino acid residues from the protein sequence. Typically, no more thanabout from 2 to 6 residues are deleted at any one site within theprotein molecule. These variants ordinarily are prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the protein,thereby producing DNA encoding the variant, and thereafter expressingthe DNA in recombinant cell culture. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, for example M13 primer mutagenesis and PCR mutagenesis. Aminoacid substitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 10 amino acid residues; and deletions willrange about from 1 to 30 residues. Deletions or insertions preferablyare made in adjacent pairs, i.e. a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof may be combined to arrive at a final construct. The mutationsmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNAstructure. Substitutional variants are those in which at least oneresidue has been removed and a different residue inserted in its place.Such substitutions generally are made in accordance with the followingTables 1 and 2 and are referred to as conservative substitutions.

TABLE 1 Amino Acid Abbreviations Amino Acid Abbreviations alanine AlaAallosoleucine AIle arginine ArgR asparagine AsnN aspartic acid AspDcysteine CysC glutamic acid GluE glutamine GlnK glycine GlyG histidineHisH isolelucine IleI leucine LeuL lysine LysK phenylalanine PheFproline ProP pyroglutamic Glu acidp serine SerS threonine ThrT tyrosineTyrY tryptophan TrpW valine ValV

TABLE 2 Amino Acid Substitutions Original Residue Exemplary ConservativeSubstitutions, others are known in the art. Alaser Arglys, gln Asngln;his Aspglu Cysser Glnasn, lys Gluasp Glypro Hisasn; gln Ileleu; valLeuile; val Lysarg; gln; MetLeu; ile Phemet; leu; tyr Serthr ThrserTrptyr Tyrtrp; phe Valile; leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table2, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in the proteinproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine, in this case, (e) by increasing the number of sites forsulfation and/or glycosylation.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variationsof each explicitly disclosed sequence are included within the mosaicpolypeptides provided herein.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco pp 79-86[1983]), acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl.

It is understood that one way to define the variants and derivatives ofthe disclosed proteins herein is through defining the variants andderivatives in terms of homology/identity to specific known sequences.For example, SEQ ID NO: 63 sets forth a particular sequence of DEFB1 andSEQ ID NO: 45 sets forth a particular sequence of PAX2. Specificallydisclosed are variants of these and other proteins herein disclosedwhich have at least, 70% or 75% or 80% or 85% or 90% or 95% homology tothe stated sequence. Those of skill in the art readily understand how todetermine the homology of two proteins. For example, the homology can becalculated after aligning the two sequences so that the homology is atits highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment.

It is understood that the description of conservative mutations andhomology can be combined together in any combination, such asembodiments that have at least 70% homology to a particular sequencewherein the variants are conservative mutations.

As this specification discusses various proteins and protein sequencesit is understood that the nucleic acids that can encode those proteinsequences are also disclosed. This would include all degeneratesequences related to a specific protein sequence, i.e. all nucleic acidshaving a sequence that encodes one particular protein sequence as wellas all nucleic acids, including degenerate nucleic acids, encoding thedisclosed variants and derivatives of the protein sequences. Thus, whileeach particular nucleic acid sequence may not be written out herein, itis understood that each and every sequence is in fact disclosed anddescribed herein through the disclosed protein sequence. For example,one of the many nucleic acid sequences that can encode the DEFB1 proteinsequence set forth in SEQ ID NO: 63 is set forth in SEQ ID NO: 64. It isalso understood that while no amino acid sequence indicates whatparticular DNA sequence encodes that protein within an organism, whereparticular variants of a disclosed protein are disclosed herein, theknown nucleic acid sequence that encodes that protein in the particularDEFB1 from which that protein arises is also known and herein disclosedand described.

It is understood that there are numerous amino acid and peptide analogswhich can be incorporated into the disclosed compositions. For example,there are numerous D amino acids or amino acids which have a differentfunctional substituent then the amino acids shown in Table 1 and Table2. The opposite stereo isomers of naturally occurring peptides aredisclosed, as well as the stereo isomers of peptide analogs. These aminoacids can readily be incorporated into polypeptide chains by chargingtRNA molecules with the amino acid of choice and engineering geneticconstructs that utilize, for example, amber codons, to insert the analogamino acid into a peptide chain in a site specific way (Thorson et al.,Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion inBiotechnology, 3:348-354 (1992); Ibba, Biotechnology & GeneticEnginerring Reviews 13:197-216 (1995), Cahill et al., TIBS,14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba andHennecke, Bio/technology, 12:678-682 (1994) all of which are hereinincorporated by reference at least for material related to amino acidanalogs).

Molecules can be produced that resemble peptides, but which are notconnected via a natural peptide linkage. For example, linkages for aminoacids or amino acid analogs can include CH₂NH—, —CH₂S—, —CH₂—CH₂—,—CH═CH— (cis and, trans), —COCH₂—, —CH(OH)CH₂—, and —CHH₂SO— (These andothers can be found in Spatola, A. F. in Chemistry and Biochemistry ofAmino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker,New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1,Issue 3, Peptide Backbone Modifications (general review); Morley, TrendsPharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res14:177-185 (1979) (—CH₂NH—, CH₂CH₂—); Spatola et al. Life Sci38:1243-1249 (1986) (—CH H₂—S); Hann J. Chem. Soc Perkin Trans. I307-314 (1982) (—CH—CH—, cis and trans); Almquist et al. J. Med. Chem.23:1392-1398 (1980) (—COCH₂—); Jennings-White et al. Tetrahedron Lett23:2533 (1982) (—COCH₂—); Szelke et al. European Appin, EP 45665 CA(1982): 97:39405 (1982) (—CH(OH)CH₂—); Holladay et al. Tetrahedron. Lett24:4401-4404 (1983) (—C(OH)CH₂—); and Hruby Life Sci 31:189-199 (1982)(—CH₂—S—); each of which is incorporated herein by reference. Aparticularly preferred non-peptide linkage is —CH₂NH—. It is understoodthat peptide analogs can have more than one atom between the bond atoms,such as b-alanine, g-aminobutyric acid, and the like.

Amino acid analogs and analogs and peptide analogs often have enhancedor desirable properties, such as, more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc.), altered specificity (e.g., abroad-spectrum of biological activities), reduced antigenicity, andothers.

D-amino acids can be used to generate more stable peptides, because Damino acids are not recognized by peptidases and such. Systematicsubstitution of one or more amino acids of a consensus sequence with aD-amino acid of the same type (e.g., D-lysine in place of L-lysine) canbe used to generate more stable peptides. Cysteine residues can be usedto cyclize or attach two or more peptides together. This can bebeneficial to constrain peptides into particular conformations. (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference).

Pharmaceutical Carriers/Delivery of Pharmaceutical Products

As described above, the compositions can also be administered in vivo ina pharmaceutically acceptable carrier. By “pharmaceutically acceptable”is meant a material that is not biologically or otherwise undesirable,i.e., the material may be administered to a subject, along with thenucleic acid or vector, without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art.

The compositions may be administered orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, topically or the like,including topical intranasal administration or administration byinhalant. As used herein, “topical intranasal administration” meansdelivery of the compositions into the nose and nasal passages throughone or both of the nares and can comprise delivery by a sprayingmechanism or droplet mechanism, or through aerosolization of the nucleicacid or vector. Administration of the compositions by inhalant can bethrough the nose or mouth via delivery by a spraying or dropletmechanism. Delivery can also be directly to any area of the respiratorysystem (e.g., lungs) via intubation. The exact amount of thecompositions required will vary from subject to subject, depending onthe species, age, weight and general condition of the subject, theseverity of the allergic disorder being treated, the particular nucleicacid or vector used, its mode of administration and the like. Thus, itis not possible to specify an exact amount for every composition.However, an appropriate amount can be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth” and otherantibody conjugated liposomes (including lipid mediated drug targetingto colonic carcinoma), receptor mediated targeting of DNA through cellspecific ligands, lymphocyte directed tumor targeting, and highlyspecific therapeutic retroviral targeting of murine glioma cells invivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

Pharmaceutically Acceptable Carriers

The compositions, including DEFB1, DEFB1-encoding nucleic acids,oligonucleotide inhibitors of PAX2 binding, can be used therapeuticallyin combination with a pharmaceutically acceptable carrier.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutionis preferably from about 5 to about 8, and more preferably from about 7to about 7.5. Further carriers include sustained release preparationssuch as semipermeable matrices of solid hydrophobic polymers containingthe antibody, which matrices are in the form of shaped articles, e.g.,films, liposomes or microparticles. It will be apparent to those personsskilled in the art that certain carriers may be more preferabledepending upon, for instance, the route of administration andconcentration of composition being administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions may also includeone or more active ingredients such as antimicrobial agents,antiinflammatory agents, anesthetics, and the like.

The pharmaceutical composition may be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration may be topically (includingophthalmically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection. The disclosedantibodies can be administered intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like.

Preservatives and other additives may also be present such as, forexample, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

Therapeutic Uses

Effective dosages and schedules for administering the compositions maybe determined empirically, and making such determinations is within theskill in the art. The dosage ranges for the administration of thecompositions are those large enough to produce the desired effect inwhich the symptoms disorder are effected. The dosage should not be solarge as to cause adverse side effects, such as unwantedcross-reactions, anaphylactic reactions, and the like. Generally, thedosage will vary with the age, condition, sex and extent of the diseasein the patient, route of administration, or whether other drugs areincluded in the regimen, and can be determined by one of skill in theart. The dosage can be adjusted by the individual physician in the eventof any counterindications. Dosage can vary, and can be administered inone or more dose administrations daily, for one or several days.Guidance can be found in the literature for appropriate dosages forgiven classes of pharmaceutical products. For example, guidance inselecting appropriate doses for antibodies can be found in theliterature on therapeutic uses of antibodies, e.g., Handbook ofMonoclonal Antibodies, Ferrone et al., eds., Noges Publications, ParkRidge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies inHuman Diagnosis and Therapy, Haber et al., eds., Raven Press, New York(1977) pp. 365-389. A typical daily dosage of the oligonucleotide usedalone might range from about 1 μg/kg to up to 100 mg/kg of body weightor more per day, depending on the factors mentioned above. In a morespecific example, 5 to 7 mg/kg/day can be used. This is appropriate fori.v. administration and higher dosages, up to about 150 mg/m²/day s.c.are appropriate. This can be administered daily for a week in from 1 to24 courses. See for example A Phase I Pharmacokinetic and BiologicalCorrelative Study of Oblimersen Sodium (Genasense, G3139), an AntisenseOligonucleotide to the Bcl-2 mRNA, and of Docetaxel in Patients withHormone-Refractory Prostate Cancer, Clinical Cancer Research, Vol. 10,5048-5057, Aug. 1, 2004, incorporated herein for it's teaching ofdosages for oligonucleotides.

Following administration of a disclosed composition for treating cancer,the efficacy of the therapeutic composition can be assessed in variousways well known to the skilled practitioner. For instance, one ofordinary skill in the art will understand that a composition, DEFB1,DEFB1-encoding nucleic acid, inhibitor of PAX2, disclosed herein isefficacious in treating cancer in a subject by observing that thecomposition reduces tumor load or prevents a further increase in tumorload. Methods of assessing tumor load are known in the art

The compositions that inhibit the interactions between PAX2 and theDEFB1 promoter can be administered prophylactically to patients orsubjects who are at risk for cancer.

Other molecules that interact with PAX2 to inhibit its interaction withthe DEFB1 promoter can be delivered in ways similar to those describedfor the pharmaceutical products.

The disclosed compositions and methods can also be used for example astools to isolate and test new drug candidates for a variety of relateddiseases. Thus, a method of identifying inhibitors of the binding ofPAX2 to the DEFB1 promoter is provided. The method can comprisecontacting a system that expresses DEFB1 with a putative inhibitor inthe presence and/or absence of PAX2 to determine whether there is aninhibitory effect on this interaction.

Compositions Identified by Screening with DisclosedCompositions/Combinatorial Chemistry

Combinatorial Chemistry

The disclosed compositions can be used as targets for any combinatorialtechnique to identify molecules or macromolecular molecules thatinteract with the disclosed compositions in a desired way. Alsodisclosed are the compositions that are identified through combinatorialtechniques or screening techniques in which the compositions disclosedas the PAX2 sequence or portions thereof (e.g., PAX2 DNA-bindingdomain), are used as the target in a combinatorial or screeningprotocol.

It is understood that when using the disclosed compositions incombinatorial techniques or screening methods, molecules, such asmacromolecular molecules, will be identified that have particulardesired properties such as inhibition or stimulation or the targetmolecule's function. The molecules identified and isolated when usingthe disclosed compositions, such as, DEFB1 or PAX2, are also disclosed.Thus, the products produced using the combinatorial or screeningapproaches that involve the disclosed compositions are also consideredherein disclosed.

It is understood that the disclosed methods for identifying moleculesthat inhibit the interactions between, for example, DEFB1 promoter andPAX2 can be performed using high through put means. For example,putative inhibitors can be identified using Fluorescence ResonanceEnergy Transfer (FRET) to quickly identify interactions. The underlyingtheory of the techniques is that when two molecules are close in space,ie, interacting at a level beyond background, a signal is produced or asignal can be quenched. Then, a variety of experiments can be performed,including, for example, adding in a putative inhibitor. If the inhibitorcompetes with the interaction between the two signaling molecules, thesignals will be removed from each other in space, and this will cause adecrease or an increase in the signal, depending on the type of signalused. This decrease or increasing signal can be correlated to thepresence or absence of the putative inhibitor. Any signaling means canbe used. For example, disclosed are methods of identifying an inhibitorof the interaction between any two of the disclosed moleculescomprising, contacting a first molecule and a second molecule togetherin the presence of a putative inhibitor, wherein the first molecule orsecond molecule comprises a fluorescence donor, wherein the first orsecond molecule, typically the molecule not comprising the donor,comprises a fluorescence acceptor; and measuring Fluorescence ResonanceEnergy Transfer (FRET), in the presence of the putative inhibitor andthe in absence of the putative inhibitor, wherein a decrease in FRET inthe presence of the putative inhibitor as compared to FRET measurementin its absence indicates the putative inhibitor inhibits binding betweenthe two molecules. This type of method can be performed with a cellsystem as well.

Combinatorial chemistry includes but is not limited to all methods forisolating small molecules or macromolecules that are capable of bindingeither a small molecule or another macromolecule, typically in aniterative process. Proteins, oligonucleotides, and sugars are examplesof macromolecules. For example, oligonucleotide molecules with a givenfunction, catalytic or ligand-binding, can be isolated from a complexmixture of random oligonucleotides in what has been referred to as “invitro genetics” (Szostak, TIBS 19:89, 1992). One synthesizes a largepool of molecules bearing random and defined sequences and subjects thatcomplex mixture, for example, approximately 10¹⁵ individual sequences in100 μg of a 100 nucleotide RNA, to some selection and enrichmentprocess. Through repeated cycles of affinity chromatography and PCRamplification of the molecules bound to the ligand on the column,Ellington and Szostak (1990) estimated that 1 in 10¹⁰ RNA moleculesfolded in such a way as to bind a small molecule dyes. DNA moleculeswith such ligand-binding behavior have been isolated as well (Ellingtonand Szostak, 1992; Bock et al, 1992). Techniques aimed at similar goalsexist for small organic molecules, proteins, antibodies and othermacromolecules known to those of skill in the art. Screening sets ofmolecules for a desired activity whether based on small organiclibraries, oligonucleotides, or antibodies is broadly referred to ascombinatorial chemistry. Combinatorial techniques are particularlysuited for defining binding interactions between molecules and forisolating molecules that have a specific binding activity, often calledaptamers when the macromolecules are nucleic acids.

There are a number of methods for isolating proteins which either havede novo activity or a modified activity. For example, phage displaylibraries have been used to isolate numerous peptides that interact witha specific target. (See for example, U.S. Pat. Nos. 6,031,071;5,824,520; 5,596,079; and 5,565,332 which are herein incorporated byreference at least for their material related to phage display andmethods relate to combinatorial chemistry)

A preferred method for isolating proteins that have a given function isdescribed by Roberts and Szostak (Roberts R. W. and Szostak J. W. Proc.Natl. Acad. Sci. USA, 94(23)12997-302 (1997). This combinatorialchemistry method couples the functional power of proteins and thegenetic power of nucleic acids. An RNA molecule is generated in which apuromycin molecule is covalently attached to the 3′-end of the RNAmolecule. An in vitro translation of this modified RNA molecule causesthe correct protein, encoded by the RNA to be translated. In addition,because of the attachment of the puromycin, a peptdyl acceptor whichcannot be extended, the growing peptide chain is attached to thepuromycin which is attached to the RNA. Thus, the protein molecule isattached to the genetic material that encodes it. Normal in vitroselection procedures can now be done to isolate functional peptides.Once the selection procedure for peptide function is completetraditional nucleic acid manipulation procedures are performed toamplify the nucleic acid that codes for the selected functionalpeptides. After amplification of the genetic material, new RNA istranscribed with puromycin at the 3′-end, new peptide is translated andanother functional round of selection is performed. Thus, proteinselection can be performed in an iterative manner just like nucleic acidselection techniques. The peptide which is translated is controlled bythe sequence of the RNA attached to the puromycin. This sequence can beanything from a random sequence engineered for optimum translation (i.e.no stop codons etc.) or it can be a degenerate sequence of a known RNAmolecule to look for improved or altered function of a known peptide.The conditions for nucleic acid amplification and in vitro translationare well known to those of ordinary skill in the art and are preferablyperformed as in Roberts and Szostak (Roberts R. W. and Szostak J. W.Proc. Natl. Acad. Sci. USA, 94(23)12997-302 (1997)).

Another preferred method for combinatorial methods designed to isolatepeptides is described in Cohen et al. (Cohen B. A., et al., Proc. Natl.Acad. Sci. USA 95(24):14272-7 (1998)). This method utilizes and modifiestwo-hybrid technology. Yeast two-hybrid systems are useful for thedetection and analysis of protein:protein interactions. The two-hybridsystem, initially described in the yeast Saccharomyces cerevisiae, is apowerful molecular genetic technique for identifying new regulatorymolecules, specific to the protein of interest (Fields and Song, Nature340:245-6 (1989)). Cohen et al., modified this technology so that novelinteractions between synthetic or engineered peptide sequences could beidentified which bind a molecule of choice. The benefit of this type oftechnology is that the selection is done in an intracellularenvironment. The method utilizes a library of peptide molecules thatattached to an acidic activation domain.

Using methodology well known to those of skill in the art, incombination with various combinatorial libraries, one can isolate andcharacterize those small molecules or macromolecules, which bind to orinteract with the desired target. The relative binding affinity of thesecompounds can be compared and optimum compounds identified usingcompetitive binding studies, which are well known to those of skill inthe art.

Techniques for making combinatorial libraries and screeningcombinatorial libraries to isolate molecules which bind a desired targetare well known to those of skill in the art. Representative techniquesand methods can be found in but are not limited to U.S. Pat. Nos.5,084,824, 5,288,514, 5,449,754, 5,506,337, 5,539,083, 5,545,568,5,556,762, 5,565,324, 5,565,332, 5,573,905, 5,618,825, 5,619,680,5,627,210, 5,646,285, 5,663,046, 5,670,326, 5,677,195, 5,683,899,5,688,696, 5,688,997, 5,698,685, 5,712,146, 5,721,099, 5,723,598,5,741,713, 5,792,431, 5,807,683, 5,807,754, 5,821,130, 5,831,014,5,834,195, 5,834,318, 5,834,588, 5,840,500, 5,847,150, 5,856,107,5,856,496, 5,859,190, 5,864,010, 5,874,443, 5,877,214, 5,880,972,5,886,126, 5,886,127, 5,891,737, 5,916,899, 5,919,955, 5,925,527,5,939,268, 5,942,387, 5,945,070, 5,948,696, 5,958,702, 5,958,792,5,962,337, 5,965,719, 5,972,719, 5,976,894, 5,980,704, 5,985,356,5,999,086, 6,001,579, 6,004,617, 6,008,321, 6,017,768, 6,025,371,6,030,917, 6,040,193, 6,045,671, 6,045,755, 6,060,596, and 6,061,636.

Combinatorial libraries can be made from a wide array of molecules usinga number of different synthetic techniques. For example, librariescontaining fused 2,4-pyrimidinediones (U.S. Pat. No. 6,025,371)dihydrobenzopyrans (U.S. Pat. Nos. 6,017,768and 5,821,130), amidealcohols (U.S. Pat. No. 5,976,894), hydroxy-amino acid amides (U.S. Pat.No. 5,972,719) carbohydrates (U.S. Pat. No. 5,965,719),1,4-benzodiazepin-2,5-diones (U.S. Pat. No. 5,962,337), cyclics (U.S.Pat. No. 5,958,792), biaryl amino acid amides (U.S. Pat. No. 5,948,696),thiophenes (U.S. Pat. No. 5,942,387), tricyclic Tetrahydroquinolines(U.S. Pat. No. 5,925,527), benzofurans (U.S. Pat. No. 5,919,955),isoquinolines (U.S. Pat. No. 5,916,899), hydantoin and thiohydantoin(U.S. Pat. No. 5,859,190), indoles (U.S. Pat. No. 5,856,496),imidazol-pyrido-indole and imidazol-pyrido-benzothiophenes (U.S. Pat.No. 5,856,107) substituted 2-methylene-2,3-dihydrothiazoles (U.S. Pat.No. 5,847,150), quinolines (U.S. Pat. No. 5,840,500), PNA (U.S. Pat. No.5,831,014), containing tags (U.S. Pat. No. 5,721,099), polyketides (U.S.Pat. No. 5,712,146), morpholino-subunits (U.S. Pat. Nos. 5,698,685 and5,506,337), sulfamides (U.S. Pat. No. 5,618,825), and benzodiazepines(U.S. Pat. No. 5,288,514).

Screening molecules similar to the disclosed siRNA molecules forinhibition of PAX2 suppression of DEFB1 expresson is a method ofisolating desired compounds.

Molecules isolated which can either be competitive inhibitors ornon-competitive inhibitors.

In another embodiment the inhibitors are non-competitive inhibitors. Onetype of non-competitive inhibitor will cause allosteric rearrangements.

As used herein combinatorial methods and libraries included traditionalscreening methods and libraries as well as methods and libraries used initerative processes.

Computer Assisted Drug Design

The disclosed compositions can be used as targets for any molecularmodeling technique to identify either the structure of the disclosedcompositions or to identify potential or actual molecules, such as smallmolecules, which interact in a desired way with the disclosedcompositions. The nucleic acids, peptides, and related moleculesdisclosed herein can be used as targets in any molecular modelingprogram or approach.

It is understood that when using the disclosed compositions in modelingtechniques, molecules, such as macromolecular molecules, will beidentified that have particular desired properties such as inhibition orstimulation of the target molecule's function. The molecules identifiedand isolated when using the disclosed compositions, such as, SEQ IDNO:1, are also disclosed. Thus, the products produced using themolecular modeling approaches that involve the disclosed compositions,such as, SEQ ID NO:1, are also considered herein disclosed.

Thus, one way to isolate molecules that bind a molecule of choice isthrough rational design. This is achieved through structural informationand computer modeling. Computer modeling technology allows visualizationof the three-dimensional atomic structure of a selected molecule and therational design of new compounds that will interact with the molecule.The three-dimensional construct typically depends on data from x-raycrystallographic analyses or NMR imaging of the selected molecule. Themolecular dynamics require force field data. The computer graphicssystems enable prediction of how a new compound will link to the targetmolecule and allow experimental manipulation of the structures of thecompound and target molecule to perfect binding specificity. Predictionof what the molecule-compound interaction will be when small changes aremade in one or both requires molecular mechanics software andcomputationally intensive computers, usually coupled with user-friendly,menu-driven interfaces between the molecular design program and theuser.

Examples of molecular modeling systems are the CHARMm and QUANTAprograms, Polygen Corporation, Waltham, Mass. CHARMm performs the energyminimization and molecular dynamics functions. QUANTA performs theconstruction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive withspecific proteins, such as Rotivinen, et al., 1988 Acta PharmaceuticaFennica 97, 159-166; Ripka, New Scientist 54-57 (Jun. 16, 1988);McKinaly and Rossmann, 1989 Annu. Rev. Pharmacol. Toxiciol. 29, 111-122;Perry and Davies, QSAR: Quantitative Structure-Activity Relationships inDrug Design pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989Proc. R. Soc. Lond. 236, 125-140 and 141-162; and, with respect to amodel enzyme for nucleic acid components, Askew, et al., 1989 J. Am.Chem. Soc. 111, 1082-1090. Other computer programs that screen andgraphically depict chemicals are available from companies such asBioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga, Ontario,Canada, and Hypercube, Inc., Cambridge, Ontario. Although these areprimarily designed for application to drugs specific to particularproteins, they can be adapted to design of molecules specificallyinteracting with specific regions of DNA or RNA, once that region isidentified.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichalter substrate binding or enzymatic activity.

Computer Readable Mediums

It is understood that the disclosed nucleic acids and proteins can berepresented as a sequence consisting of the nucleotides of amino acids.There are a variety of ways to display these sequences, for example thenucleotide guanosine can be represented by G or g. Likewise the aminoacid valine can be represented by Val or V. Those of skill in the artunderstand how to display and express any nucleic acid or proteinsequence in any of the variety of ways that exist, each of which isconsidered herein disclosed. Specifically contemplated herein is thedisplay of these sequences on computer readable mediums, such as,commercially available floppy disks, tapes, chips, hard drives, compactdisks, and video disks, or other computer readable mediums. Alsodisclosed are the binary code representations of the disclosedsequences. Those of skill in the art understand what computer readablemediums. Thus, computer readable mediums on which the nucleic acids orprotein sequences are recorded, stored, or saved.

Disclosed are computer readable mediums comprising the sequences andinformation regarding the sequences set forth herein. Also disclosed arecomputer readable mediums comprising the sequences and informationregarding the sequences set forth.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in C or is atambient temperature, and pressure is at or near atmospheric.

Example I Human Beta Defensin-1 is Cytotoxic to Late-Stage ProstateCancer and Plays a Role in Prostate Cancer Tumor Immunity Abstract

DEFB1 was cloned into an inducible expression system to examine whateffect it had on normal prostate epithelial cells, as well as androgenreceptor positive (AR⁺) and androgen receptor negative (AR⁻) prostatecancer cell lines. Induction of DEFB1 expression resulted in a decreasein cellular growth in AR⁻ cells DU145 and PC3, but had no effect on thegrowth of the AR⁺ prostate cancer cells LNCaP. DEFB1 also caused rapidinduction of caspase-mediated apoptosis. Data presented here are thefirst to provide evidence of its role in innate tumor immunity andindicate that its loss contributes to tumor progression in prostatecancer.

Materials and Methods Cell Lines

The cell lines DU145 were cultured in DMEM medium, PC3 were grown in F12medium, and LNCaP were grown in RPMI medium (Life Technologies, Inc.,Grand Island, N.Y.). Growth media for all three lines was supplementedwith 10% (v/v) fetal bovine serum (Life Technologies). The hPrEC cellswere cultured in prostate epithelium basal media (Cambrex Bio Science,Inc., Walkersville, Md.). All cell lines were maintained at 37° C. and5% CO₂.

Tissue Samples and Laser Capture Microdissection

Prostate tissues obtained from consented patients that underwent radicalprostatectomy were acquired through the Hollings Cancer Center tumorbank in accordance with an Institutional Review. Board-approvedprotocol. This included guidelines for the processing, sectioning,histological characterization, RNA purification and PCR amplification ofsamples. Following pathologic examination of frozen tissue sections,laser capture microdissection (LCM) was performed to ensure that thetissue samples assayed consisted of pure populations of benign prostatecells. For each tissue section analyzed, LCM was performed at threedifferent regions containing benign tissue and the cells collected werethen pooled.

Cloning of DEFB1 Gene

DEFB1 cDNA was generated from RNA by reverse transcription-PCR. The PCRprimers were designed to contain ClaI and KpnI restriction sites. DEFB1PCR products were restriction digested with ClaI and KpnI and ligatedinto a TA cloning vector. The TA/DEFB1 vector was then transfected intoE. coli by heat shock and individual clones were selected and expanded.Plasmids were isolated by Cell Culture DNA Midiprep (Qiagen, Valencia,Calif.) and sequence integrity verified by automated sequencing. TheDEFB1 gene fragment was then ligated into the pTRE2 digested with ClaIand KpnI, which served as an intermediate vector for orientationpurposes. Then the pTRE2/DEFB1 construct was digested with ApaI and KpnIto excise the DEFB1 insert, which was ligated into pIND vector of theEcdysone Inducible Expression System (Invitrogen, Carlsbad, Calif.) alsodouble digested with ApaI and KpnI. The construct was again transfectedinto E. coli and individual clones were selected and expanded. Plasmidswere isolated and sequence integrity of pIND/DEFB1 was again verified byautomated sequencing.

Transfection

Cells (1×10⁶) were seeded onto 100-mm Petri dishes and grown overnight.Then the cells were co-transfected using Lipofectamine 2000 (Invitrogen,Carlsbad, Calif.) with 1 μg of pVgRXR plasmid, which expresses theheterodimeric ecdysone receptor, and 1 μg of the pIND/DEFB1 vectorconstruct or empty pIND control vector in Opti-MEM media (LifeTechnologies, Inc., Grand Island, N.Y.).

RNA Isolation and Quantitative RT-PCR

In order to verify DEFB1 protein expression in the cells transfectedwith DEFB1 construct, RNA was collected after a 24 hour induction periodwith Ponasterone A (Pon A). Briefly, total RNA was isolated using the SVTotal RNA Isolation System (Promega, Madison, Wis.) from approximately1×10⁶ cells harvested by trypsinizing. Here, cells were lysed and totalRNA was isolated by centrifugation through spin columns. For cellscollected by LCM, total RNA was isolated using the PicoPure RNAIsolation Kit (Arcturus Biosciences, Mt. View, Calif.) following themanufacturer's protocol. Total RNA (0.5 μg per reaction) from bothsources was reverse transcribed into cDNA utilizing random primers(Promega). AMV Reverse Transcriptase II enzyme (500 units per reaction;Promega) was used for first strand synthesis and Tfl DNA Polymerase forsecond strand synthesis (500 units per reaction; Promega) as per themanufacturer's protocol. In each case, 50 pg of cDNA was used perensuing PCR reaction. Two-step QRT-PCR was performed on cDNA generatedusing the MultiScribe Reverse Transcripatase from the TaqMan ReverseTranscription System and the SYBR Green PCR Master Mix (AppliedBiosystems).

The primer pair for DEFB1 (Table 3) was generated from the publishedDEFB1 sequence (GenBank Accession No. U50930)¹⁰. Forty cycles of PCRwere performed under standard conditions using an annealing temperatureof 56° C. In addition, β-actin (Table 3) was amplified as a housekeepinggene to normalize the initial content of total cDNA. DEFB1 expressionwas calculated as the relative expression ratio between DEFB1 andβ-actin and was compared in cells lines induced and uninduced for DEFB1expression, as well as LCM benign prostatic tissue. As a negativecontrol, QRT-PCR reactions without cDNA template were also performed.All reactions were run three times in triplicate.

MTT Cell Viability Assay

To examine the effects of DEFB1 on cell growth, metabolic3-[4,5-dimethylthiazol-2yl]-2,5 diphenyl tetrazolium bromide (MTT)assays were performed. PC3, DU145 and LNCaP cells co-transfected withpVgRXR plasmid and pIND/DEFB1 construct or empty pIND vector were seededonto a 96-well plate at 1-5×10³ cells per well. Twenty-four hours afterseeding, fresh growth medium was added containing 10 μM Ponasterone Adaily to induce DEFB1 expression for 24-, 48- and 72 hours after whichthe MTT assay was performed according to the manufacturer's instructions(Promega). Reactions were performed three times in triplicate.

Flow Cytometry

PC3 and DU145 cells co-transfected with the DEFB1 expression system weregrown in 60-mm dishes and induced for 12, 24, and 48 hours with 10 μMPonasterone A. Following each incubation period, the medium wascollected from the plates (to retain any detached cells) and combinedwith PBS used to wash the plates. The remaining attached cells wereharvested by trypsinization and combined with the detached cells andPBS. The cells were then pelleted at 4° C. (500×g) for 5 min, washedtwice in PBS, and resuspended in 100 ul of 1× Annexin binding buffer(0.1 M Hepes/NaOH at pH 7.4, 1.4 M NaCl, 25 mM CaCl₂) containing 5 μl ofAnnexin V-FITC and 5 μl of PI. The cells were incubated at RT for 15 minin the dark, then diluted with 400 μl of 1× Annexin binding buffer andanalyzed by FACscan (Becton Dickinson, San Jose, Calif.). All reactionswere performed three times.

Microscopic Analysis

Cell morphology was analyzed by phase contrast microscopy. DU145, PC3and LNCaP cells containing no vector, empty plasmid or DEFB1 plasmidwere seeded onto 6 well culture plates (BD Falcon, USA). The followingday plasmid-containing cells were induced for a period of 48 h withmedia containing 10 μM Ponasterone A, while control cells received freshmedia. The cells were then viewed under an inverted Zeiss IM 35microscope (Carl Zeiss, Germany). Phase contrast pictures of a field ofcells were obtained using the SPOT Insight Mosaic 4.2 camera (DiagnosticInstruments, USA). Cells were examined by phase contrast microscopyunder 32× magnification and digital images were stored as uncompressedTIFF files and exported into Photoshop CS software (Adobe Systems, SanJose, Calif.) for image processing and hard copy presentation.

Caspase Detection

Detection of caspase activity in the prostate cancer cell lines wasperformed using APO LOGIX™ Carboxyfluorescin Caspase detection kit (CellTechnology, Mountain View, Calif.). Active caspases were detectedthrough the use of a FAM-VAD-FMK inhibitor that irreversibly binds toactive caspases. Briefly, DU145 and PC3 cells (1.5-3×10⁵) containing theDEFB1 expression system were plated in 35 mm glass bottom microwelldishes (Matek, Ashland, Mass.) and treated for 24 hours with media onlyor with media containing PonA as previously described. Next, 10 μl of a30× working dilution of carboxyfluorescein labeled peptide fluoromethylketone (FAM-VAD-FMK) was added to 300 μl of media and added to each 35mm dish. Cells were then incubated for 1 hour at 37° C. under 5% CO₂.Then, the medium was aspirated and the cells were washed twice with 2 mlof a 1× Working dilution Wash Buffer. Cells were viewed underdifferential interference contrast (DIC) or under laser excitation at488 nm. The fluorescent signal was analyzed using a confocal microscope(Zeiss LSM 5 Pascal) and a 63× DIC oil lens with a Vario 2 RGB LaserScanning Module.

Statistical Analysis

Statistical differences were evaluated using the Student's t-test forunpaired values. P values were determined by a two-sided calculation,and a P value of less than 0.05 was considered statisticallysignificant.

Results DEFB1 Expression in Prostate Tissue and Cell Lines

DEFB1 expression levels were measured by QRT-PCR in benign and malignantprostatic tissue, hPrEC prostate epithelial cells and DU145, PC3 andLNCaP prostate cancer cells. DEFB1 expression was detected in all of thebenign clinical samples. The average amount of DEFB1 relative expressionwas 0.0073. In addition, DEFB1 relative expression in hPrEC cells was0.0089. There was no statistical difference in DEFB1 expression detectedin the benign prostatic tissue samples and hPrEC (FIG. 1A). Analysis ofthe relative DEFB1 expression levels in the prostate cancer cell linesrevealed significantly lower levels in DU145, PC3 and LNCaP. As afurther point of reference, relative DEFB1 expression was measured inthe adjacent malignant section of prostatic tissue from patient #1215.There were no significant differences in the level of DEFB1 expressionobserved in the three prostate cancer lines compared to malignantprostatic tissue from patient #1215 (FIG. 1B). In addition, expressionlevels in all four samples were close to the no template negativecontrols which confirmed little to no endogenous DEFB1 expression (datanot shown). QRT-PCR was also performed on the prostate cancer cell linestransfected with the DEFB1 expression system. Following a 24 hourinduction period, relative expression levels were 0.01360 in DU145,0.01503 in PC3 and 0.138 in LNCaP. Amplification products were verifiedby gel electrophoresis.

QRT-PCR was performed on LCM tissues regions containing benign, PIN andcancer. DEFB1 relative expression was 0.0146 in the benign regioncompared to 0.0009 in the malignant region (FIG. 1C.). This represents a94% decrease which again demonstrates a significant down-regulation ofexpression. Furthermore, analysis of PIN revealed that DEFB1 expressionlevel was 0.044 which was a 70% decrease. Comparing expression inpatient #1457 to the average expression level found in benign regions ofsix other patients (FIG. 1A.) revealed a ratio of 1.997 representingalmost twice as much expression (FIG. 1D.). However, the expressionratio was 0.0595 in PIN and was 0.125 in malignant tissue compared toaverage expression levels in benign tissue.

DEFB1 Causes Cell Membrane Permeability and Ruffling

Induction of DEFB1 in the prostate cancer cell lines resulted in asignificant reduction in cell number in DU145 and PC3, but had no effecton cell proliferation in LNCaP (FIG. 2). As a negative control, cellproliferation was monitored in all three lines containing empty plasmid.There were no observable changes in cell morphology in DU145, PC3 orLNCaP cells following the addition of PonA. In addition, DEFB1 inductionresulted in morphological changes in both DU145 and PC3. Here cellsappeared more rounded and exhibited membrane ruffling indicative of celldeath. Apoptotic bodies were also present in both lines.

Expression of DEFB1 Results in Decreased Cell Viability

The MTT assay showed a reduction in cell viability by DEFB1 in PC3 andDU145 cells, but no significant effect on LNCaP cells (FIG. 3). After 24hours, relative cell viability was 72% in DU145 and 56% in PC3. Analysis48 hours after induction revealed 49% cell viability in DU145 and 37%cell viability in PC3. After 72 hours of DEFB1 expression resulted in44% and 29% relative cell viability in DU145 and PC3 cells,respectively.

DEFB1 Causes Rapid Caspase-Mediated Apoptosis in Late-Stage ProstateCancer Cells

In order to determine whether the effects of DEFB1 on PC3 and DU145 werecytostatic or cytotoxic, FACS analysis was performed. Under normalgrowth conditions, more than 90% of PC3 and DU145 cultures were viableand non-apoptotic (lower left quadrant) and did not stain with annexin Vor PI (FIG. 4). After inducing DEFB1 expression in PC3 cells, the numberof apoptotic cells (lower and upper right quadrants) totaled 10% at 12hours, 20% at 24 hours, and 44% at 48 hours. For DU145 cells, the numberof apoptotic cells totaled 12% after 12 hours, 34% at 24 hours, and 59%after 48 hours of induction. There was no increase in apoptosis observedin cells containing empty plasmid following induction with PonA (datanot shown).

Caspase activity was determined by confocal laser microscopic analysis(FIG. 5). DU145 and PC3 cell were induced for DEFB1 expression andactivity was monitored based on the binding of green fluoresingFAM-VAD-FMK to caspases in cells actively undergoing apoptosis. Analysisof cells under DIC showed the presence of viable control DU145 (A), PC3(E) and LNCaP (I) cells at 0 hours. Excitation by the confocal laser at488 nm produced no detectable green staining which indicates no caspaseactivity in DU145 (B), PC3 (F) or LNCaP (J). Following induction for 24hours, DU145 (C), PC3 (G) and LNCaP (K) cells were again visible underDIC. Confocal analysis under fluorescence revealed green staining inDU145 (D) and PC3 (H) cell indicating caspase activity. However, therewas no green staining in LNCaP (L), indicating no induction of apoptosisby DEFB1.

Conclusion

To assess its functional role, the DEFB1 gene was cloned into theecdysone inducible expression system and examined its effect on prostatecancer cells. The present data demonstrate DEFB1 cytotoxic activityagainst late-stage androgen receptor negative hormone refractoryprostate cancer cells. In conclusion, this study provides the functionalrole of DEFB1 in prostate cancer. Furthermore, these findings show thatDEFB1 is part of an innate immune system involved in tumor immunity.Data presented here demonstrate that DEFB1 expressed at physiologicallevels is cytotoxic to AR⁻ hormone refractory prostate cancer cells, butnot to AR+ hormone sensitive prostate cancer cell nor to normal prostateepithelial cells. Given that DEFB1 is constitutively expressed in normalprostate cells without cytotoxicity, it may be that late-stage AR⁻prostate cancer cells possess distinct phenotypic characteristics thatrender them sensitive to DEFB1 cytotoxicity. Thus, DEFB1 is a viabletherapeutic agent for the treatment of late-stage prostate cancer.

Example II siRNA Mediated Knockdown of PAX2 Expression Results inProstate Cancer Cell Death Independent of p53 Status Abstract

This example examines the effects of inhibiting PAX2 expression by RNAinterference in prostate cancer cells which differ in p53 gene status.These results demonstrate that the inhibition of PAX2 results in celldeath irrespective of p53 status, indicating that there are additionaltumor suppressor genes or cell death pathways inhibited by PAX2 inprostate cancer.

Materials and Methods Cell Lines

The cell lines PC3, DU145 and LNCaP were obtained from the American TypeCulture Collection (Rockville, Md., USA). PC3 cell were grown in F-12media, DU145 in DMEM, and LNCaP in RPMI all supplemented with 10% (v/v)fetal bovine serum. Cell were maintained at 37° C. in 5% CO₂.

siRNA Silencing of PAX2

In order to achieve efficient gene silencing, a pool of fourcomplementary short interfering ribonucleotides (siRNAs) targeting humanPAX2 mRNA (Accession No. NM_(—)003989.1), were synthesized (DharmaconResearch, Lafayette, Colo., USA). A second pool of four siRNAs were usedas an internal control to test for the specificity of PAX2 siRNAs. Twoof the sequences synthesized target the GL2 luciferase mRNA (AccessionNo. X65324), and two were non-sequence-specific (Table 4). For annealingof siRNAs, 35 M of single strands were incubated in annealing buffer(100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesiumacetate) for 1 min at 90° C. followed by 1 h incubation at 37° C.

Western Analysis

Briefly, cells were harvested by trypsinization and washed twice withPBS. Lysis buffer was prepared according to the manufacturer'sinstructions (Sigma), and was then added to the cells. Following a 15minute incubation period at 4° C. on an orbital shaker, cell lysate werethen collected and centrifuged for 10 minutes at 12000×g to pelletcellular debris. The protein-containing supernatant were then collectedand quantitated. Next, 25 μg protein extract was loaded onto an 8-16%gradient SDS-PAGE (Novex). Following electrophoresis, proteins weretransferred to PVDF membranes, and then blocked with 5% nonfat dry milkin TTBS (0.05% Tween 20 and 100 mM Tris-Cl) for 1 hour. Blots were thenprobed with rabbit anti-Pax2 primary antibody (Zymed, San Francisco,Calif.) at a 1:2000 dilution. After washing, the membranes wereincubated with anti-rabbit antibody conjugated to horseradish peroxidase(HRP) (dilution 1:5000; Sigma), and signal detection was visualizedusing chemilluminescence reagents (Pierce) on an Alpha InnotechFluorchem 8900. As a control, blots were stripped and reprobed withmouse anti-β-actin primary antibody (1:5000; Sigma-Aldrich) andHRP-conjugated anti-mouse secondary antibody (1:5000; Sigma-Aldrich) andsignal detection was again visualized.

Phase Contrast Microscopy

The effect of PAX2 knock-down on cell growth was analyzed by phasecontrast microscopy. Here, 1-2×10⁴ cells were seeded onto 6 well cultureplates (BD Falcon, USA). The following day cells were treated with mediaonly, negative control non-specific siRNA or PAX2 siRNA and allowed toincubate for six days. The cells were then viewed under an invertedZeiss IM 35 microscope (Carl Zeiss, Germany) at 32× magnification. Phasecontrast pictures of a field of cells were obtained using the SPOTInsight Mosaic 4.2 camera (Diagnostic Instruments, USA).

MTT Cytotoxicity Assay

DU145, PC3 and LNCaP cells (1×10⁵) were transfected with 0.5 μg of thePAX2 siRNA pool or control siRNA pool using Codebreaker transfectionreagent according to the manufacturer's protocol (Promega). Next, cellsuspensions were diluted and seeded onto a 96-well plate at 1-5×10³cells per well and allowed to grow for 2-, 4- or 6 days. After culture,cell viability was determined by measuring the conversion of3-[4,5-dimethylthiazol-2yl]-2,5 diphenyl tetrazolium bromide, MTT(Promega), to a colored formazan product. Absorbance was read at 540 nmon a scanning multiwell spectrophotometer.

Pan-Caspase Detection

Detection of caspase activity in the prostate cancer cell lines wasperformed using APO LOGIX™ Carboxyfluorescin Caspase detection kit (CellTechnology, Mountain View, Calif.). Active caspases were detectedthrough the use of a FAM-VAD-FMK inhibitor that irreversibly binds toactive caspases. Briefly, cells (1-2×10⁴) onto 35 mm glass bottommicrowell dishes (Matek, Ashland, Mass.) and treated with media only orPAX2 siRNA as previously described. Next, 10 μl of a 30× workingdilution of carboxyfluorescein labeled peptide fluoromethyl ketone(FAM-VAD-FMK) was added to 300 μl of media and added to each 35 mm dish.Cells were then incubated for 1 hour at 37° C. under 5% CO₂. Then, themedium was aspirated and the cells were washed twice with 2 ml of a 1×Working dilution Wash Buffer. Cells were viewed under differentialinterference contrast (DIC) or under laser excitation at 488 nm. Thefluorescent signal was analyzed using a confocal microscope (Zeiss LSM 5Pascal) and a 63× DIC oil lens with a Vario 2 RGB Laser Scanning Module.

Quantitative Real-time RT-PCR

Quantitative real-time RT-PCR was performed in order to verify geneexpression after PAX2 siRNA treatment in PC3, DU145 and LNCaP celllines. Total RNA was isolated using the SV Total RNA Isolation System(Promega). Briefly, approximately. 1×10⁶ cells were harvested bytrypsinizing, and rinsed in PBS. Cells were then lysed and total RNA wasisolated by centrifugation through spin columns. Total RNA (0.5 μg perreaction) was reverse transcribed into cDNA utilizing Oligo (dT) 15primer (Promega) and AMV Reverse Transcriptase II enzyme (500 units perreaction; Promega) for first strand synthesis and Tfl DNA Polymerase forfor second strand synthesis (500 units per reaction; Promega) as per themanufacturers' protocol, with identical control samples treated withoutRT enzyme. Typically, 50 pg of each cDNA was used per ensuing PCRreaction Two-step QRT-PCR was performed on cDNA generated using theMultiScribe Reverse Transcripatase from the TaqMan Reverse TranscriptionSystem and the SYBR Green PCR Master Mix (PE Biosystems). The primerpairs for BAX, BID and BAD were generated from the published sequences(Table 3). Reactions were performed in MicroAmp Optical 96-well ReactionPlate (PE Biosystems). Forty cycles of PCR were performed under standardconditions using an annealing temperature of 60° C. Quantification wasdetermined by the cycle number where exponential amplification began(threshold value) and averaged from the values obtained from thetriplicate repeats. There was an inverse relationship between messagelevel and threshold value. In addition, GAPDH was used as a housekeepinggene to normalize the initial content of total cDNA. Gene expression wascalculated as the relative expression ratio between the pro-apoptoticgenes and GAPDH. All reactions were carried out in triplicate.

Results

siRNA Inhibition of PAX2 Protein

In order to confirm that the siRNA effective targeted the PAX2 mRNA,Western Analysis was performed to monitor PAX2 protein expression levelsover a six day treatment period. Cells were given a single round oftransfection with the pool of PAX2 siRNA. The results confirmed specifictargeting of PAX2 mRNA by showing knock-down of PAX2 protein by day fourin DU145 (FIG. 6 a) and by day six in PC3 (FIG. 6 b).

Knock-Down of PAX2 Inhibit Prostate Cancer Cell Growth

Cells were analyzed following a six day treatment period with mediaonly, negative control non-specific siRNA or PAX2 siRNA (FIG. 7). DU145(a), PC3 (d) and LNCaP (g) cells all reached at least 90% confluency inthe culture dishes containing media only. Treatment of DU145 (b), PC3(e) and LNCaP (h) with negative control non-specific siRNA had no effecton cell growth, and cells again reached confluency after six days.However, treatment with PAX2 siRNA resulted in a significant decrease incell number. DU145 cells were approximately 15% confluent (c) and PC3cells were only 10% confluent (f). LNCaP cell were 5% confluentfollowing siRNA treatment.

Cytotoxicity Assays

Cell viability was measured after two-, four-, and six-day exposuretimes, and is expressed as a ratio of the 570-630 nm absorbance oftreated cells divided by that of the untreated control cells (FIG. 8).Relative cell viability following 2 days of treatment was 77% in LNCaP,82% in DU145 and 78% in PC3. After four days, relative cell viabilitywas 46% in LNCaP, 53% in DU145 and 63% in PC3. After six days oftreatment, relative cell viability decreased to 31% in LNCaP, 37% inPC3, and was 53% in DU145. As negative controls, cell viability wasmeasured in after a six day treatment period with negative controlnon-specific siRNA or transfection reagent alone. For both conditions,there was no statistically significant change in cell viability comparedto normal growth media (data not shown).

Pan-Caspase Detection

Caspase activity was detected by confocal laser microscopic analysis.DU145, PC3 and LNCaP cells were treated with PAX2 siRNA and activity wasmonitored based on the binding of FAM-labeled peptide to caspases incells actively undergoing apoptosis which will fluoresce green. Analysisof cells with media only under DIC shows the presence of viable DU145(A), PC3 (E) and LNCaP (I) cells at 0 hours (FIG. 9). Excitation by theconfocal laser at 488 nm produced no detectable green staining whichindicates no caspase activity in untreated DU145 (B), PC3 (F) or LNCaP(J). Following four days of treatment with PAX2 siRNA, DU145 (C), PC3(G) and LNCaP (K) cells were again visible under DIC. Underfluorescence, the treated DU145 (D), PC3 (H) and LNCaP (L) cellspresented green staining indicating caspase activity.

Effect of Pax2 Inhibition on Pro-Apoptotic Factors

DU145, PC3 and LNCaP cells were treated with siRNA against PAX2 for sixdays and expression of pro-apoptotic genes dependent and independent ofp53 transcription regulation were measured to monitor cell deathpathways. For BAX, there was a 1.81-fold increase in LNCaP, a 2.73-foldincrease in DU145, and a 1.87-fold increase in PC3 (FIG. 10 a).Expression levels of BID increased by 1.38-fold in LNCaP and 1.77-foldin DU145 (FIG. 10 b). However, BID expression levels decreased by1.44-fold in PC3 following treatment (FIG. 10 c). Analysis of BADrevealed a 2.0-fold increase in expression in LNCaP, a 1.38-foldincrease in DU145, and a 1.58-fold increase in PC3.

Conclusion

Despite significant advances in cancer therapy there is still littleprogress in the treatment of advanced disease. Successful drug treatmentof prostate cancer requires the use of therapeutics with specificeffects on target cells while maintaining minimal clinical effects onthe host. The goal of cancer therapy is to trigger tumor-selective celldeath. Therefore, understanding the mechanisms in such death is criticalin determining the efficacy of a specific treatment.

The dependency of prostate cancer cell survival on PAX2 expression isshown here. In order to distinguish between death observed in thep53-expressing cell line LNCaP, the p53-mutated line DU145, and thep53-null line PC3 downstream events that follow p53 activation as aresult of PAX2 knock-down were examined. Caspase activity was detectedin all three lines indicative of the initiation of programmed celldeath. With this, changes in the expression of pro-apoptotic genes wereexamined. Here, BAX expression was upregulated in all three cell linesindependent of p53 status. The expression of pro-apoptotic factor BADwas increased in all three lines following PAX2 inhibition. Followingtreatment with PAX2 siRNA, BID expression was increased in LNCaP andDU145, but actually decreased in PC3. This indicates that cell deathobserved in prostate cancer is influenced by but is not dependent on p53expression. The initiation of apoptosis in prostate cancer cells throughdifferent cell death pathways irrespective of p53 status indicates thatPAX2 inhibits other tumor suppressors

Example III Inhibition of PAX2 Oncogene Results in DEFB1-Mediated Deathof Prostate Cancer Cells Abstract

The identification of tumor-specific molecules that serve as targets forthe development of new cancer drugs is considered to be a major goal incancer research. Example I demonstrated that there is a high frequencyof DEFB1 expression loss in prostate cancer, and that induction of DEFB1expression results in rapid apoptosis in androgen receptornegative-stage prostate cancer. These data show that DEFB1 plays a rolein prostate tumor suppression. In addition, given that it is a naturallyoccurring component of the immune system of normal prostate epithelium,DEFB1 is expected to be a viable therapeutic agent with little to noside effects. Example II demonstrated that inhibition of PAX2 expressionresults in prostate cancer cell death independent of p53. These dataindicate that there is an addition pro-apoptotic factor or tumorsuppressor that is inhibited by PAX2. In addition, the data show thatthe oncogenic factor PAX2, which is over-expressed in prostate cancer,is a transcriptional repressor of DEFB1. The purpose of this study is todetermine if DEFB1 loss of expression is due to aberrant expression ofthe PAX2 oncogene, and whether inhibiting PAX2 results in DEFB1-mediatedcell death.

The data show that loss of DEFB1 expression occurs at thetranscriptional level. Furthermore, computational analysis of the DEFB1promoter revealed the presence of a GTTCC (SEQ ID NO: 2) DNA bindingsite for the PAX2 transcriptional repressor next to the DEFB1 TATA box(FIG. 1). The results presented here show that PAX2 and DEFB1 exhibitseveral attributes of suitable cancer targets, including a role in thesuppression of cell death. Therefore, DEFB1 plays a role in tumorimmunity and its expression is modulated through therapeuticdown-regulation of the PAX2 oncogene.

Materials and Methods RNA Isolation and Quantitative RT-PCR

In order to verify changes in DEFB1 expression levels RNA was collectedafter 4 days of PAX2 siRNA treatment. Briefly, total RNA was isolatedusing the SV Total RNA Isolation System (Promega, Madison, Wis.) fromapproximately 1×10⁶ cells harvested by trypsinizing. Here, cells werelysed and total RNA was isolated by centrifugation through spin columns.Total RNA (0.5 μg per reaction) from both sources was reversetranscribed into cDNA utilizing random primers (Promega). AMV ReverseTranscriptase II enzyme (500 units per reaction; Promega) was used forfirst strand synthesis and Tfl DNA Polymerase for second strandsynthesis (500 units per reaction; Promega) as per the manufacturer'sprotocol. In each case, 50 pg of cDNA was used per ensuing PCR reaction.Two-step QRT-PCR was performed on cDNA generated using the MultiScribeReverse Transcripatase from the TaqMan Reverse Transcription System andthe SYBR Green PCR Master Mix (Applied Biosystems).

The primer pair for DEFB1 was generated from the published DEFB1sequence (Accession No. U50930). Forty cycles of PCR were performedunder standard conditions using an annealing temperature of 56° C. Inaddition, GAPDH was amplified as a housekeeping gene to normalize theinitial content of total cDNA. DEFB1 expression was calculated as therelative expression ratio between DEFB1 and GAPDH and was compared incells lines before and after siRNA knock-down of PAX2 expression. Allreactions were run three times in triplicate.

Generation of the DEFB1 Reporter Construct

The pGL3 luciferase reporter plasmid was used to monitor DEFB1 reporteractivity. Here, a region 160 bases upstream of the DEFB1 transcriptioninitiation site and included the DEFB1 TATA box. The region alsoincluded the GTTCC (SEQ ID NO: 2) sequence which is necessary for PAX2binding. The PCR primers were designed to contain KpnI and NheIrestriction sites. The DEFB1 promoter PCR products were restrictiondigested KpnI and NheI and ligated into a similary restriction digestedpGL3 plasmid (FIG. 2). The constructs were transfected into E. coli andindividual clones were selected and expanded. Plasmids were isolated andsequence integrity of the DEFB1/pGL3 construct was verified by automatedsequencing.

Luciferase Reporter Assay

Here, 1 μg of the DEFB1 reporter construct or the control pGL3 plasmidwas transfected into 1×10⁶ DU145 cells. Next, 0.5×10³ cells were seededonto each well of a 96-well plate and allowed to grow overnight. Thenfresh medium was added containing PAX2 siRNA or media only and the cellswere incubated for 48 hours. Luciferase was detected by the BrightGlokit accourding to the manufacturer's protocol (Promega) and the plateswere read on a Veritas automated 96-well luminometer. Promoter activitywas expressed as relative luminescence.

Analysis of Membrane Permeability

Acridine orange (AO)/ethidium bromide (EtBr) dual staining was performedto identify changes in cell membrane integrity, as well as apoptoticcells by staining the condensed chromatin. AO stains viable cells aswell as early apoptotic cells, whereas EtBr stains late stage apoptoticcells that have lost membrane permeability. Briefly, cells were seededinto 2 chamber culture slides (BD Falcon, USA). Cells transfected withempty pIND plasmid/pvgRXR or pIND DEFB1/pvgRXR were induced for 24 or 48h with media containing 10 μM Ponasterone A. Control cells were providedfresh media at 24 and 48 h. In order to determine the effect of PAX2inhibition on membrane integrity, separate culture slides containingDU145, PC3 and LNCaP were treated with PAX2 siRNA and incubated for 4days. Following this, cells were washed once with PBS and stained with 2ml of a mixture (1:1) of AO (Sigma, USA) and EtBr (Promega, USA) (5ug/ml) solution for 5 min. Following staining, the cells were againwashed with PBS. Fluorescence was viewed by a Zeiss LSM 5 Pascal Vario 2Laser Scanning Confocal Microscope (Carl Zeiss Jena, Germany). Theexcitation color wheel contain BS505-530 (green) and LP560 (red) filterblocks which allowed for the separation of emitted green light from AOinto the green channel and red light from EtBr into the red channel. Thelaser power output and gain control settings within each individualexperiment were identical between control and DEFB1 induced cells. Theexcitation was provided by a Kr/Ar mixed gas laser at wavelengths of 543nm for AO and 488 nm for EtBr. Slides were analyzed under 40×magnification and digital images were stored as uncompressed TIFF filesand exported into Photoshop CS software (Adobe Systems, San Jose,Calif.) for image processing and hard copy presentation.

ChIP Analysis of PAX2

Chromatin immunoprecipitation (ChIP) allows the identification ofbinding sites for DNA-binding proteins based upon in vivo occupancy of apromoter by a transcription factor and enrichment of transcriptionfactor bound chromatin by immunoprecipitation (66). A modification ofthe protocol described by the Farnham laboratory ((67, 68) was used;also on line at http://mcardle.oncology.wisc.edu/farnham/). The DU145and PC3 cell lines over-expresses the PAX2 protein, but does not expressDEFB1. Cells were incubated with PBS containing 1.0% formaldehyde for 10minutes to crosslink proteins to DNA. Samples were then sonicated toyield DNA with an average length of 600 bp. Sonicated chromatinprecleared with Protein A Dynabeads was incubated with PAX2-specificantibody or “no antibody” control [isotype-matched control antibodies].Washed immunoprecipitates were then collected. After reversal of thecrosslinks, DNA was analyzed by PCR using promoter-specific primers todetermine whether DEFB1 is represented in the PAX2-immunoprecipitatedsamples. Primers were designed to amplify the 160 by region immediatelyupstream of the DEFB1 mRNA start site which contained the DEFB1 TATA boxand the functional GTTCC (SEQ ID NO: 2) PAX2 recognition site. For thesestudies, positive controls included PCR of an aliquot of the inputchromatin (prior to immunoprecipitation, but crosslinks reversed). Allsteps were performed in the presence of protease inhibitors.

Results

siRNA Inhibition of PAX2 Increases DEFB1 Expression

QRT-PCR analysis of DEFB1 expression before siRNA treatment revealedrelative expression levels of 0.00097 in DU145, 0.00001 in PC3, and0.00004 LNCaP (FIG. 13). Following siRNA knock-down of PAX2, relativeexpression was 0.03294 (338-fold increase) in DU145, 0.00020 (22.2-foldincrease) in PC3 and 0.00019 (4.92-fold increase) in LNCaP. As anegative control, the human prostate epithelial cell line (hPrEC) whichis PAX2 null, revealed expression levels at 0.00687 before treatment and0.00661 following siRNA treatment confirming no statistical change inDEFB1 expression.

DEFB1 Causes Cell Membrane Permeability

Membrane integrity was monitored by confocal analysis (FIG. 14). Here,intact cells stain green due to AO which is membrane permeable. Inaddition, cells with compromised plasma membranes would stain red byEtBr which is membrane impermeable. Here, uninduced DU145 (A) and PC3(D) cells stained positively with AO and emitted green color, but didnot stain with EtBr. However, DEFB1 induction in both DU145 (B) and PC3(E) resulted in the accumulation of EtBr in the cytoplasm at 24 hoursindicated by the red staining. By 48 hours, DU145 (C) and PC3 (F)possessed condensed nuclei and appeared yellow, which was due to thepresence of both green and red staining resulting from the accumulationof AO and EtBr, respectively.

Inhibition of PAX2 Results in Membrane Permeability

Cells were treated with PAX2 siRNA for 4 days and membrane integrity wasmonitored again by confocal analysis (FIG. 15). Here, both DU145 (B) andPC3 (E) possessed condensed nuclei and appeared yellow. However, LNCaPcells' cytoplasm and nuclei remained green following siRNA treament.Also red staining at the cell periphery indicates the maintenance ofcell membrane integrity. These findings indicate that the inhibition ofPAX2 results in specifically DEFB1-mediated cell death in DU1145 andPC3, but not LNCaP cells. Death observed in LNCaP (refer to Chapter II)is due to the transactivation of the existing wild-type p53 in LNCapfollowing PAX2 inhibition.

siRNA Inhibition of PAX2 Increases DEFB1 Promoter Activity

Analysis of DEFB1 promoter activity in DU 145 cells containing theDEFB1/pGL3 construct revealed a 2.65 fold increase in relative lightunits following 48 hours of treatment compared to untreated cells (FIG.16). In PC3 cells, there was a 3.78-fold increase in relative lightunits compared to untreated cells.

PAX2 Binds to the DEFB1 Promoter

ChIP analysis was performed on DU145 and PC3 cells to determine if thePAX2 transcriptional repressor is bound to the DEFB1 promoter (FIG. 17).Lane 1 contains a 100 by molecular weight marker. Lane 2 is a positivecontrol representing 160 by region of the DEFB1 promoter amplified fromDU145 before cross-linking and immunoprecipitation. Lane 3 is a negativecontrol representing PCR performed without DNA. Lane 4 and 5 arenegative controls representing PCR from immunoprecipitations performedwith IgG from cross-linked DU145 and PC3, respectively. PCRamplification of 25 pg of DNA (lane 6 and 8) and 50 pg of DNA (lane 7and 9) immunoprecitipated with anti-PAX2 antibody after crosslinkingshow 160 by promoter fragment in DU145 and PC3, respectively.

Conclusion

The present novel data are the first to disclose the role of DEFB1 inprostate cancer tumor immunity. The data also show that the oncogenicfactor PAX2 suppresses DEFB1 expression. One of the hallmarks ofdefensin cytotoxicity is the disruption of membrane integrity. Thepresent results show that ectopic expression of DEFB1 in prostate cancercells results in a loss of membrane potential due to compromised cellmembranes. The same phenomenon is observed after inhibiting PAX2 proteinexpression. ChIP analysis was also performed and confirmed that PAX2 isbound to the DEFB1 promoter resulting in the repression of DEFB1expression. Therefore, suppression of PAX2 expression or function,results in the re-establishment of DEFB1 expression and subsequentlyDEFB 1-mediated cell death. Also, the present data establish the utilityof DEFB1 as a directed therapy for prostate cancer treatment throughinnate immunity.

Example IV Expression of DEFB1 Results in Tumor Shrinkage

The anti-tumoral ability of DEFB1 is evaluated by injecting tumor cellsthat overexpress DEFB1 into nude mice. DEFB1 is cloned into pBI-EGFPvector, which has a bidirectional tetracycline responsible promoter.Tet-Off Cell lines are generated by transfecting pTet-Off into DU145,PC3 and LNCaP cells and selecting with G418. The pBI-EGFP-DEFB1 plasmidis co-transfected with pTK-Hyg into the Tet-off cell lines and selectedwith hygromycin. Only single-cell suspensions with a viability of >90%are used. Each animal receives approximately 500,000 cells administeredsubcutaneously into the right flank of female nude mice. There are twogroups, a control group injected with vector only clones and a groupinjected with the DEFB1 over-expressing clones. 35 mice are in eachgroup as determined by a statistician. Animals are weighed twice weekly,tumor growth monitored by calipers and tumor volumes determined usingthe following formula: volume=0.5×(width)2×length. All animals aresacrificed by CO₂ overdose when tumor size reaches 2 mm³ or 6 monthsfollowing implantation; tumors are excised, weighed and stored inneutral buffered formalin for pathological examination. Differences intumor growth between the groups are descriptively characterized throughsummary statistics and graphical displays. Statistical significance isevaluated with either the t-test or non-parametric equivalent.

Example V Expression of PAX2 siRNA Results in Up-Regulation of DEFB1Expression and Tumor Shrinkage In Vivo

Hairpin PAX2 siRNA template oligonucleotides utilized in the in vitrostudies are utilized to examine the effect of the up-regulation of DEFB1expression in vivo. The sense and antisense strand (see Table 4) areannealed and cloned into pSilencer 2.1 U6 hygro siRNA expression vector(Ambion) under the control of the human U6 RNA pol III promoter. Thecloned plasmid is sequenced, verified and transfected into PC3, Du145,and LNCap cell lines. Scrambled shRNA is cloned and used as a negativecontrol in this study. Hygromycin resistant colonies are selected, cellsare introduced into the mice subcutaneously and tumor growth ismonitored as described above.

Example VI Small Molecule Inhibitors of PAX2 Binding Results inUp-Regulation of DEFB1 Expression and Tumor Shrinkage In Vivo

The DNA recognition sequence for PAX2 binding resides in the DEFB1promoter between nucleotides −75 and −71 [+1 refers to thetranscriptional start site]. Short oligonucleotides complementary to thePAX2 DNA-binding domain are provided. Examples of such oligonucleotidesinclude the 20-mer and 40-mer oligonucleotides containing the GTTCC (SEQID NO: 2) recognition sequence provided below. These lengths wererandomly selected, and other lengths are expected to be effective asblockers of binding. As a negative control, oligonicleotides with ascrambled sequence (CTCTG) (SEQ ID NO: 22) were designed to verifyspecificity. The oligonucleotides are transfected into the prostatecancer cells and the HPrEC cells with lipofectamine reagent orCodebreaker transfection reagent (Promega, Inc). In order to confirmDNA-protein interactions, double stranded oligonucleotides will belabeled with [32P] dCTP and electrophoretic mobility shift assays areperformed. In addition, DEFB1 expression is monitored by QRT-PCR andWestern analysis following treatment with oligonucleotides. Finally,cell death is detected by MTT assay and flow cytometry as previouslydescribed.

Recognition Sequence #1: (SEQ ID NO: 18) CTCCCTTCAGTTCCGTCGACRecognition Sequence #2: (SEQ ID NO: 19) CTCCCTTCACCTTGGTCGAC ScrambleSequence #1: (SEQ ID NO: 23) CTCCCTTCACTCTGGTCGAC Recognition Sequence#3: (SEQ ID NO: 20) ACTGTGGCACCTCCCTTCAGTTCCGTCGACGAGGTTGTGC RecognitionSequence #4: (SEQ ID NO: 21) ACTGTGGCACCTCCCTTCACCTTGGTCGACGAGGTTGTGCScramble Sequence #2: (SEQ ID NO: 24)ACTGTGGCACCTCCCTTCACTCTGGTCGACGAGGTTGTGC

Further examples of oligonucleotides of the invention include:

Recognition Sequence #1: (SEQ ID No: 25) 5′-AGAAGTTCACCCTTGACTGT-3′Recognition Sequence #2: (SEQ ID No: 26) 5′-AGAAGTTCACGTTCCACTGT-3′Scramble Sequence #1: (SEQ ID No: 27) 5′-AGAAGTTCACGCTCTACTGT-3′Recognition Sequence #3: (SEQ ID No: 28)5′-TTAGCGATTAGAAGTTCACCCTTGACTGTGGCACCTCCC-3′ Recognition Sequence #4:(SEQ ID No: 29) 5′-GTTAGCGATTAGAAGTTCACGTTCCACTGTGGCACGTCCC-3′ ScrambleSequence #2: (SEQ ID No: 30)5′-GTTAGCGATTAGAAGTTCACGCTCTACTGTGGCACCTCCC-3′

This set of alternative inhibitory oligonucleotides represents therecognition sequence (along with the CCTTG (SEQ ID NO: 1) core sequence)for the PAX2 binding domain and homeobox. These include actual sequencesfrom the DEFB1 promoter.

The PAX2 gene is required for the growth and survival of various cancercells including prostate. In addition, the inhibition of PAX2 expressionresults in cell death mediated by the innate immunity component DEFB1.Suppression of DEFB1 expression and activity is accomplished by bindingof the PAX2 protein to a GTTCC (SEQ ID NO: 2) recognition site in theDEFB1 promoter. Therefore, this pathway provides a viable therapeutictarget for the treatment of prostate cancer. In this method, thesequences bind to the PAX2 DNA binding site and block PAX2 binding tothe DEFB1 promoter thus allowing DEFB1 expression and activity. Theoligonucleotide sequences and experiment described above are examples ofand demonstrate a model for the design of additional PAX2 inhibitordrugs.

Given that the GTTCC (SEQ ID NO: 2) sequence exists in interleukin-3,interleukin-4, the insulin receptor and others, PAX2 regulates theirexpression and activity as well. Therefore the PAX2 inhibitors disclosedherein have utility in a number of other diseases including thosedirected related to inflammation including prostatitis and benignprostatic hypertrophy (BPH).

Example VII Loss of DEFB1 Expression Results in Increased TumorigenesisGeneration of Loss of Function Mice

The Cre/loxP system has been useful in elucidating the molecularmechanisms underlying prostate carcinogenesis. Here a DEFB1 Creconditional KO is used for inducible disruption within the prostate. TheDEFB1 Cre conditional KO involves the generation of a targeting vectorcontaining loxP sites flanking DEFB1 coding exons, targeted ES cellswith this vector and the generation of germline chimeric mice from thesetargeted ES cells. Heterozygotes are mated to prostate-specific Cretransgenics and heterozygous intercross is used to generateprostate-specific DEFB1 KO mice. Four genotoxic chemical compounds havebeen found to induce prostate carcinomas in rodents:N-methyl-N-nitrosourea (MNU), N-nitrosobis 2-oxopropyl. amine (BOP),3,2X-dimethyl-4-amino-biphenyl (MAB) and2-amino-1-methyl-6-phenylimidazow 4,5-bxpyridine (PhIP).DEFB1-transgenic mice are treated with these carcinogenic compounds viaintra-gastric administration or i.v. injection for prostate adenoma andadenocarcinoma induction studies. Prostate samples are studied fordifferences in tumor growth and changes gene expression thoughhistological, immunohistological, mRNA and protein analyses.

Generation of GOF Mice

For PAX2 inducible GOF mice, PAX2 GOF (bi-transgenic) and wild-type(mono-transgenic) littermates are administered doxycycline (Dox) from 5weeks of age to induce prostate-specific PAX2 expression. Briefly,PROBASIN-rtTA mono-transgenic mice (prostate cell-specific expression oftet-dependent rtTA inducer) are crossed to our PAX2 transgenic responderlines. For induction, bi-transgenic mice are fed Dox via the drinkingwater (500 mg/L freshly prepared twice a week). Initial experimentsverify low background levels, good inducibility and cell-type specificexpression of PAX2 and the EGFP reporter using transgenic founder linein bi-transgenic mice. Regarding experimental group sizes, 5-7 age- andsex-matched individuals in each group (wild-type and GOF) allow forstatistical significance. For all animals in this study, prostatetissues are collected initially at weekly intervals for analysis andcomparison, to determine carcinogenic time parameters.

PCR Genotyping, RT-PCR and qPCR

PROBASIN-rtTA transgenic mice are genotyped using the following PCRprimers and conditions: PROBASIN5 (forward) 5′-ACTGCCCATTGCCCAAACAC-3′(SEQ ID NO: 31); RTTA3 (reverse) 5′-AAAATCTTGCCAGCTTTCCCC-3′ (SEQ ID NO:32); 95° C. denaturation for 5 min, followed by 30 cycles of 95° C. for30 sec, 57° C. for 30 sec., 72° C. for 30 sec, followed by a 5 minextension at 72° C., yielding a 600 by product. PAX2 inducibletransgenic mice are genotyped using the following PCR primers andconditions: PAX2For 5′-GTCGGTTACGGAGCGGACCGGAG-3′(SEQ ID NO: 33);Rev5'IRES 5′-TAACATATAGACAAACGCACACCG-3′ (SEQ ID NO: 34); 95° C.denaturation for 5 min, followed by 34cycles of 95° C. for 30 sec, 63°C. for 30 sec, 72° C. for 30 sec, followed by a 5 min extension at 72°C., yielding a 460 by product. Immortomouse hemizygotes are be genotypedusing the following PCR primers and conditions: Immol1, 5′-GCGCTTGTGTCGCCATTGTATTC-3′ (SEQ ID NO: 35); Immol2, 5′-GTCACACCACAGAAGTAAGGTTCC-3′(SEQ ID NO: 36); 94° C. 30 sec, 58° C. 1 min, 72° C. 1 min 30 sec, 30cycles to yield a ˜1 kb transgene band. For genotyping PAX2 knockoutmice, the following PCR primers and conditions are used: PAX2 For5′-GTCGGTTACGGAGCGGACCGGAG-3′ (SEQ ID NO: 37); PAX2Rev5′-CACAGAGCATTGGCGATCTCGATGC-3′ (SEQ ID NO: 38); 94° C. 1 min, 65° C. 1min, 72° C. 30 sec, 36 cycles to yield a 280 by band.

DEFB1 Peptide Animal Studies

Six-week-old male athymic (nude) mice purchased from Charles RiverLaboratories are injected sub-cutaneously over the scapula with 10⁶viable PC3 cells. One week after injection, the animals are randomlyallocated to one of three groups—group I: control; group II:intraperitoneal injections of DEFB1, 100 μg/day, 5 days a week, forweeks 2-14; group III: intraperitoneal injections of DEFB1, 100 mg/day,5 days a week, for weeks 8-14. Animals are maintained in sterilehousing, four animals to a cage, and observed on a daily basis. At10-day intervals, the tumors are measured by using calipers, and thevolumes of the tumors are calculated by using V=(L×W2)/2.

Appendix A

-   LOCUS HUMPAX201 7331 by DNA linear PRI 28 Jan. 2002-   DEFINITION Homo sapiens paired-box protein (PAX2) gene, promoter and    exon 1.-   ACCESSION L09748 AF433639-   VERSION L09748.2 GI:18141563-   KEYWORDS .-   SEGMENT 1 of 3-   SOURCE Homo sapiens (human)    -   ORGANISM Homo sapiens Eukaryota; Metazoa; Chordata; Craniata;        Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires;        Primates; Catarrhini; Hominidae; Homo.-   REFERENCE 1 (bases 7096 to 7291)    -   AUTHORS Stapleton, P., Weith, A., Urbanek, P., Kozmik, Z. and        Busslinger, M.    -   TITLE Chromosomal localization of seven PAX genes and cloning of        a novel family member, PAX-9    -   JOURNAL Nat. Genet. 3 (4), 292-298 (1993)    -   PUBMED 7981748-   REFERENCE 2 (bases 1 to 7331)    -   AUTHORS Pfeffer, P. L., Payer, B., Reim, G., di Magliano, M. P.        and Busslinger, M.    -   TITLE The activation and maintenance of Pax2 expression at the        mid-hindbrain boundary is controlled by separate enhancers    -   JOURNAL Development 129 (2), 307-318 (2002)    -   PUBMED 11807024-   REFERENCE 3 (bases 7096 to 7291)    -   AUTHORS Stapleton, P., Weith, A., Urbanek, P., Kozmik, Z. and        Busslinger, M.    -   TITLE Direct Submission    -   JOURNAL Submitted (Jun. 12, 1993) Research Institute of        Molecular Pathology, Dr. Bohr-Gasse 7, Vienna, Austria-   REFERENCE 4 (bases 1 to 7331)    -   AUTHORS Pfeffer, P. L. and Busslinger, M.    -   TITLE Direct Submission    -   JOURNAL Submitted (Jan. 14, 2002) Research Institute of        Molecular Pathology, Dr. Bohr-Gasse 7, Vienna, Austria    -   REMARK Sequence update by submitter-   COMMENT On Jan. 14, 2002 this sequence version replaced gi:292378.

FEATURES Location/Qualifiers source 1 . . . 7331 /organism = “ Homosapiens” /mol_type = “ genomic DNA” /db_xref = “ taxon:9606” /chromosome= “ 10” /map = “ 10q22.1-q24.3” gene order(2034 . . . 7331,L09746.1:1 .. . 206, L09747.1:1 . . . >238) /gene = “ PAX2” enhancer 2034 . . . 2518/gene = “ PAX2” /note = “ distal (late) enhancer” enhancer 2850 . . .2974 /gene = “ PAX2” /note = “ intermediate (early) enhancer” enhancer3516 . . . 3898 /gene = “ PAX2” /note = “ proximal (late) enhancer” mRNAjoin(6532 . . . 7271,L09746.1:21 . . . 186, L09747.1:21 . . . >218)/gene = “ PAX2” /product = “ paired-box protein” exon 6532 . . . 7271/gene = “ PAX2” /number = 1 CDS join(7229 . . . 7271,L09746.1:21 . . .186, L09747.1:21 . . . >218) /gene = “ PAX2” /codon_start = 1 /product =“ paired-box protein” /protein_id = “ AAC41711.1” /db_xref = “GI:553607” /translation = “ MDMHCKADPFSAMHRHGGVNQLGGVFVNGRPLPDVVRQRIVELAHQGVRPCDISRQLRVSHGCVSKILGRYYETGSIKPGVIGGSKPKVATPKVVDKIAEYKRQNPTMFAWEIRDRLLAEGICDNDT VPSVSSIN” (SEQ ID NO: 39)

-   ORIGIN

(SEQ ID NO: 40)    1 ttcccccttt ccangagggc ctaatccgtt gcgcgcgcgcacgcggacac acacacacac   61 acacacacac acacacacac acacacggcc cccatagccaccgcaactct cagcagcagn  121 ncctagctcc tctgacccga ggccccaaga cggcgggcacaggaacccct gggacgtcct  181 ggctccaggc tggacgtagg cggaggtggc aggagtggacaaacccaggc gggtcccacg  241 acgccccttt cctcgggtct ctccttgttt cagccagccgctctcgcccc tggtcccctc  301 ttccctgcgt tagggtcctt tgtctccagc cacctcgcagcctgtccccg cctcggcggc  361 cctgcccttt gggcctccca gatctctctg gcgggtccccctgccttacc agctcccggc  421 tgtggcgcgc tcttcgcctg ctcctcacat ncacacagctgctgggagag gaggaaggaa  481 aggcggncgc gccgcggatg gatccgagac ggtagatttggtgccggctc gcaaactctg  541 ggaaacttaa ngccggttct tccgcccctc tncaactatgnccagcgcgg cccggtcgcg  601 cgcgctcacc ccgcggggac cctttccttt tcctgtatttcggctgcggc tgtttcgctt  661 cctctggtct cccagccttt ggagtggctt ccctggccctgcactccgtt ccctttcggc  721 cgcccccggc tgtcgcctgc ccccaccctc cgcaggtcccacggtcgcgg cggcgatgac  781 tgtggaggta acgccgggga cgtcctgggt cagcctgcaccgtctccctc gaccacagcc  841 cgatgaggcc gcgggctccg ggccggctgc taagagagttaatcattact tcgccagcga  901 cactcagcct ccccttccga ctctctcgcc cggcctaggggaggagggga ggggacagct  961 ggccaggtgg ggacttcggc ttcgcacaaa ccagcctcttcaggcctccc agagacaggt 1021 ggtggcttct cagttccctc ggcaactctc taaggtcctctttcttcccc tcctgtctct 1081 ccctccttcg agcctcctcc cagccaggcc tctccccaccgtctcctgtc cgctctggct 1141 ttgactgatt aactgcaggt cctgggagaa ccaactttctttgtttggaa ccggaccgga 1201 cgggatttcc ttccctaggt ctccgccaat gggccagctcctcccgacgg ttttggcgga 1261 ctggctgaag aggaccgcgc ctgaggccac aattaacccggctgttggtg gtggtggttg 1321 gggggtgggc agtgaggaat ttaaccgatc ctctagcagctgcgctggtg cagttgggag 1381 gggggtgcag gaagtgggaa tggaggagtg gcaggaggtatagacagagg gaagaacgat 1441 aaacctggac aggtgtggca tagccaatag aaggggaaacaaaataaaac aggaaggcgg 1501 cgcggggagg aatccccagt aacctttata ggattgaagttgggtggaaa acgccacctc 1561 ctgccctacc ttagcactca gatccctcct ttacctctttgtgaaagggt aagagttcag 1621 aaagctggcc atttactcca taatctacta gagaaatgtctgggtttgca aaatgcctat 1681 tgattagctc catggagtag acaagacagg cgtaattatccccattttac aggtgagaaa 1741 actgagtctc aaagaagcaa agggactgtg tatgtagtggctgtcacttt ttcctgtagg 1801 ctgtggggtg agtggcccct ttagctgtgc agaggtccatgggtatctag ggaggcggta 1861 caggctgtgt ccaggtctga gccagaagta ccagggcctcacggggctcc tagccctttt 1921 agcttgttct ctgttggaca ggaccttcac tcttactctctagacctgct ggctgggttt 1981 ctcccagctt cgctattttt tcagttccct agtagagtggcccatgggcg gtagccacct 2041 ggctggcccg tgccactaag aggcagcttt ggtggccaagtggcttgcat tgttgttgct 2101 cctcaaaggg cctgtgaagg gctgggcagg tcgcaaagacctcttgtgag gggaaagcta 2161 gattaaaggg ggtaaggatc ctggaggata aaggccaagcacgtgcgcct ggactccaca 2221 ggaccaacag accgagcggg cggggccngc tgggagtcaggccccccggg cttcacgcag 2281 ggagcccaaa tattgggaac aaaagcagga aaagaagagtgagagcagga gggagggagg 2341 gagcgaggaa gcagaaatta gggggtctta gatgaaaaaaaaaagaaagt agctttaggg 2401 ggaatgtgct gtggagtgtg aaattgcagc ccatggtgctccatattgta ccagaagctc 2461 ttccaaaaaa aaaaaaaaaa accatcctcc aacgtgaccagagggccagg cagggggaag 2521 ggcggggaga gaatggggag gaggaggggg aaaggccgggcaggagccgg tcaggccttt 2581 ctgcggaagg ggctggggtg taagtttcgg ctccctgggatctgacagcc gagggtatgc 2641 gccctggggt gcgccgggac ccagagggcg agtgagcctcggttggtcgg ctctggagtt 2701 cggttgtcag aagaactttt atttttcttt ttggtggtgacttctaaaag tgggaataat 2761 ccagaaatga agctcagctg cggagctgca gctctgttctccctctctcc cctgcctttc 2821 tgcttctctt cccttcggac tacttttctc cccttggttctaaatagctt tttcccctct 2881 gaactttaat gcatttaatt tggtccgcgc tgtggggagcatttcctggg gagatgcatt 2941 taatttcgga atttctaatc ccctccctca gaccccggtcctagctcccc tagccgctcc 3001 ccgggaagtg gaaggaggaa ggcaggtccc ggccacgggggaggggcgcg gctgggatgc 3061 tcccgcggcc ccctccgtct caccaaggct cagccgccftcccaagctac tggaggccgg 3121 gcgcctgggc cccgggtcag ggccctgcan gaagaagagaggcaaccccc gctttctgcc 3181 ttttcttcgc ctgggcaaga aaacgctggg ccagggaactggaaaccgga aaacaggaga 3241 aagggtttnt ggaaggcanc gggagcgggt ggcagncggggcancgggca ntggactagg 3301 tctacaccgg cacttcactt ttgcacaaca tgcccagaaacgcatttgag agccctggag 3361 tcgcgcttgg cttggcttgg ggcgccggtg cgtgggtacactcgaggtcg gggtgcctat 3421 ccgccacccc gacacctaca cccagtgcag agcaggcgcggcccagccag acaaccaggc 3481 cggcagtagc tcggcctgga gggcggaggc aaggttgggggccgccaggc gcctgggcaa 3541 gcctggcagg gaagggagcc gagaaggcaa aggagccgagatccacaagg aagattnntt 3601 gggcagatca gatgcacaga ggcggctaat gaagcaaatcccgagatggg tttcagagca 3661 actccccaaa agtttatttt gcctttaaat ttccgcagggaggcgggctc cttgtttgaa 3721 gtgtaaatgc ccctaggttg gggggtggaa gggccgctttgaaaacacca gagagaaaag 3781 gttcatttag aggcggacgg gaaaagcaac caaccctgacaggtcggagc ccgggtagtg 3841 tttggggttg ggtngttttc tttctttctc tttcttttcccctttcctct tctttcttcc 3901 cttttgtgnn ttttnnttgt tttttttntn ttntttttntttaantggct ttcttgcttc 3961 cccccacccc tctactagac tctatagaag aaagagaacagaaaaggggg agtcagagga 4021 gcggccagtg actggatgaa ggccagccct tcatcctggagccccaggag aaggcagagc 4081 tttggagaaa aggggttcct aatctccagg gagcattactctttgactct ctagacccag 4141 gaatgggctg gacgctaatg gggaagcggc caggaacccggcctggcgga agagtgagtg 4201 tccagctagt gcagtgctgg gaagacgatc ccaggagcaggggggactct caggggctac 4261 ctgggaatgg gactatcaga agggtcttta ctcctcanaaggtgcatgtg aaggacaggt 4321 gtgtgaggac aacttccagc acacttggcg cattaagtccccttctctac aaaatggaaa 4381 atccttctcg cccaacatgt gaaaatgctt gttgtgggcacccacatttc atggtacttg 4441 taacatagga catgtctagc tggttctaga aaaatctgtgtctgtgtgga aggggggggg 4501 tttactcaca gctttcttcc ttcaatagtt cacacaccccgagacaaatt cctggatgac 4561 caacttggag agacctgggg caaaggttac tttagttctgagctcctcta aataaggacc 4621 ctttctcaac gttcctttca ccccagttct gggttaattacttccagtta gtgcgtgttc 4681 gtggggttgt gaggccaaag caaacccggg agcgccatctgcaggcctca agaggaagag 4741 actgacctta gaggctaggc cctgcgtctt caacctctagcccaagggaa ccaacctgcc 4801 tagccaccca agggaagtgg gataggggct gggaggggcaggcggtgagg agtgttttcc 4861 tcccagactt taccccgcag gtggattaag cttattgggctctggaggat acaggaggga 4921 gggcaaatgc caggatccca gcggacccag gccccacaggagtgagaggc tcagaacctc 4981 gtcccgctga gcctggcctg agctcctcct gaggaataagggcatcccaa aaacccgggt 5041 acaagacgcc cagtagtagt agttaggctg agtcaggcaggtgcatctct ccccatggta 5101 tctgccgccc aggctccggc cagagggagg ggagcgcgagtccgcggcgc ttccgcgggg 5161 cgcccggaac tgcagacggg ggctggagga atctcggattcgggctgcaa gagcgctgcg 5221 caagcttcgc cgagccgccc tttcgcagac ccagggaagcggggggaggg agcgaaggag 5281 ggagagagag ttaaaacatc agcttgaaag tgcccaagatgattttatta agaccgaggg 5341 gaaaattatt ttcatgaaag attctccccg gaatatttcttgtacttaac ccagttagga 5401 agacaaaggg cttctttctg cctggtgcgg tgcgagcggaccccagcgag caagggagct 5461 agtgccaaag agaactgcgg aggctccggc aggagtggggacgtccccgt ggttgcgcct 5521 cctgcgctcg ccccggatcc accgagctag cagcgggcggcgctcagccg cgtccgcagc 5581 ctcctcttct ccccagccgg ggagagccag cctcgtctcccacatcctct gccgccagcg 5641 acctgcagct ccgcactgtt tccctcccct gtacccccttcccagtcacc cgagggttca 5701 gaaaccaagt cccccggctc tcccgccatc cgctgggtcccaccgaggca ggtgggtact 5761 cgccggaggt cttcagctcg attctgaacc aagcgttctggactgcccag acccggtggg 5821 caaggggact ggggaggccc tgcgcacagt cgcgtggaacgggaggggac aagacaaact 5881 gctggacact tttccgtgga atgagaagtg gggggtgcgtgggtgggaag gtacctccgg 5941 agggaaaggc caaagggaag gaccagaaag agaggaaggaagagccggga aggaacggaa 6001 gggaactcag agccgagggt ggtggggttg gggctagggatgcgcactgg gcccggggcc 6061 gcgcggccca ggcgggcact ggccagtgga tggcagggctgggcgagtta gaactgagag 6121 cccggcttca cagcgcagcg cgctccgagg ccctctgtcgttacctgaat attcattaga 6181 ctgaccgctc tttatcctta tctaacgttt atcttatcggcgagtttcgt ttctcagtgt 6241 agttttaatc ccgggctccc attccccctc ccccggtccgctcccctccc tccctcttcc 6301 ttcgccggct gctccctccc tccctccctc ccatttctccctcccctgcc ctccccttgc 6361 cggcaccgga gtgacaggct cggggccctc ctcgccgaagctcggggctc cagcgctggc 6421 gaatcacaga gtggtggaat ctattgcctt tgtctgacaagtcatccatc tcccggcgcg 6481 gggaggggga ggaggtctgg agggggcttt gcagcttttagagagacaca caccgggagc 6541 cgaggctcca gtctccggcc gagtcttcta gcagccgcaacccacctggg gccagcccag 6601 agctgccagc gccgctcggc tccctccctc cctcccggcccttcggccgc ggcggcgtgc 6661 gcctgccttt tccgggggcg ggggcctggc ccgcgcgctcccctcccgca ggcgccacct 6721 cggacatccc cgggattgct acttctctgc caacttcgccaactcgccag cacttggaga 6781 ggcccggctc ccctcccggc gccctctgac cgcccccgccccgcgcgctc tccgaccacc 6841 gcctctcgga tgaacaggtt ccaggggagc tgagcgagtcgcctcccccg cccagcttca 6901 gccctggctg cagctgcagc gcgagccatg cgcccccagtgcaccccggc ccggcccacc 6961 gccccggggc cattctgctg accgcccagc cccgagccccgacagtggca agttgcggct 7021 actgcggttg caagctccgg ccaacccgga ggagccccagcggggagcgc agtgttgcgc 7081 cccccgcccc cgcgcgcgcc gcagcagccg ggcgttcactcatcctccct cccccaccgt 7141 ccctcccttt tctcctcaag tcctgaagtt gagtttgagaggcgacacgg cggcggcggc 7201 cgcgctgctc ccgctcctct gcctccccat ggatatgcactgcaaagcag accccttctc 7261 cgcgatgcac cgtgagtacc cgcgcccggc tcctgtcccggctcgggctc tccgtcccaa 7321 ccctgtccag t

Appendix B

-   LOCUS NM_(—)003987 4276 by mRNA linear PRI 24 Sep. 2005-   DEFINITION Homo sapiens paired box gene 2 (PAX2), transcript variant    a, mRNA.-   ACCESSION NM_(—)003987-   VERSION NM_(—)003987.2 GI:34878698-   KEYWORDS .-   SOURCE Homo sapiens (human)-   ORGANISM Homo sapiens Eukaryota; Metazoa; Chordata; Craniata;    Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires;    Primates; Catarrhini; Hominidae; Homo.-   REFERENCE 1 (bases 1 to 4276)    -   AUTHORS Yoshimura, K., Yoshida, S., Yamaji, Y., Komori, A.,        Yoshida, A., Hatae, K., Kubota, T. and Ishibashi, T.    -   TITLE De novo insG619 mutation in PAX2 gene in a Japanese        patient with papillorenal syndrome    -   JOURNAL Am. J. Ophthalmol. 139 (4), 733-735 (2005)    -   PUBMED 15808183    -   REMARK GeneRIF: Molecular genetic analysis of the PAX2 gene in        combination with renal ultrasonography can help in making an        earlier diagnosis of the disease.-   REFERENCE 2 (bases 1 to 4276)    -   AUTHORS Mazal, P. R., Stichenwirth, M., Koller, A., Blach, S.,        Haitel, A. and Susani, M.    -   TITLE Expression of aquaporins and PAX-2 compared to CD10 and        cytokeratin 7 in renal neoplasms: a tissue microarray study    -   JOURNAL Mod. Pathol. 18 (4), 535-540 (2005)    -   PUBMED 15502805    -   REMARK GeneRIF: PAX-2 is a reliable marker for clear cell renal        cell carcinomas of lower grades but not for higher grades.-   REFERENCE 3 (bases 1 to 4276)    -   AUTHORS Higashide, T., Wada, T., Sakurai, M., Yokoyama, H. and        Sugiyama, K.    -   TITLE Macular abnormalities and optic disk anomaly associated        with a new PAX2 missense mutation    -   JOURNAL Am. J. Ophthalmol. 139 (1), 203-205 (2005)    -   PUBMED 15652857    -   REMARK GeneRIF: A new PAX2 missense mutation, R71T, may cause        macular abnormalities in addition to anomalies of the optic disk        and the kidney.-   REFERENCE 4 (bases 1 to 4276)    -   AUTHORS Buttiglieri, S., Deregibus, M. C., Bravo, S., Cassoni,        P., Chiarle, R., Bussolati, B. and Camussi, G.    -   TITLE Role of Pax2 in apoptosis resistance and proinvasive        phenotype of Kaposi's sarcoma cells    -   JOURNAL J. Biol. Chem. 279 (6), 4136-4143 (2004)    -   PUBMED 14627715    -   REMARK GeneRIF: expression of Pax2 by Kaposi's sarcoma cells        correlated with an enhanced resistance against apoptotic signals        and with the proinvasive phenotype-   REFERENCE 5 (bases 1 to 4276)    -   AUTHORS Brophy, P. D., Lang, K. M. and Dressler, G. R.    -   TITLE The secreted frizzled related protein 2 (SFRP2) gene is a        target of the Pax2 transcription factor    -   JOURNAL J. Biol. Chem. 278 (52), 52401-52405 (2003)    -   PUBMED 14561758    -   REMARK GeneRIF: Pax2 protein regulates expression of secreted        frizzled related protein 2-   REFERENCE 6 (bases 1 to 4276)    -   AUTHORS Schimmenti, L. A., Manligas, G. S. and Sieving, P. A.    -   TITLE Optic nerve dysplasia and renal insufficiency in a family        with a novel PAX2 mutation, Arg115X: further ophthalmologic        delineation of the renal-coloboma syndrome    -   JOURNAL Ophthalmic Genet. 24 (4), 191-202 (2003)    -   PUBMED 14566649    -   REMARK GeneRIF: PAX2 mutation is associated with Optic nerve        dysplasia and renal insufficiency of the renal-coloboma syndrome-   REFERENCE 7 (bases 1 to 4276)    -   AUTHORS Muratovska, A., Zhou, C., He, S., Goodyer, P. and        Eccles, M. R.    -   TITLE Paired-Box genes are frequently expressed in cancer and        often required for cancer cell survival    -   JOURNAL Oncogene 22 (39), 7989-7997 (2003)    -   PUBMED 12970747    -   REMARK GeneRIF: The PAX2 gene was frequently expressed in a        panel of 406 common primary tumor tissues and endogenous PAX        gene expression is often required for the growth and survival of        cancer cells-   REFERENCE 8 (bases 1 to 4276)    -   AUTHORS Gough, S. M., McDonald, M., Chen, X. N., Korenberg, J.        R., Neri, A., Kahn, T., Eccles, M. R. and Morris, C. M.    -   TITLE Refined physical map of the human PAX2/HOX11/NFKB2 cancer        gene region at 10q24 and relocalization of the HPV6AI1 viral        integration site to 14q13.3-q21.1    -   JOURNAL BMC Genomics 4 (1), 9 (2003)    -   PUBMED 12697057-   REFERENCE 9 (bases 1 to 4276)    -   AUTHORS Hoffmeister, A., Ropolo, A., Vasseur, S., Mallo, G. V.,        Bodeker, H., Ritz-Laser, B., Dressler, G. R., Vaccaro, M. I.,        Dagorn, J. C., Moreno, S. and Iovanna, J. L.    -   TITLE The HMG-I/Y-related protein p8 binds to p300 and Pax2        trans-activation domain-interacting protein to regulate the        trans-activation activity of the Pax2A and Pax2B transcription        factors on the glucagon gene promoter    -   JOURNAL J. Biol. Chem. 277 (25), 22314-22319 (2002)    -   PUBMED 11940591    -   REMARK GeneRIF: The HMG-UY-related protein p8 binds to p300 and        Pax2 trans-activation domain-interacting protein to regulate the        trans-activation activity of the Pax2A and Pax2B transcription        factors on the glucagon gene promoter.-   REFERENCE 10 (bases 1 to 4276)    -   AUTHORS Cai, Y., Lechner, M. S., Nihalani, D., Prindle, M. J.,        Holzman, L. B. and Dressler, G. R.    -   TITLE Phosphorylation of Pax2 by the c-Jun N-terminal kinase and        enhanced Pax2-dependent transcription activation    -   JOURNAL J. Biol. Chem. 277 (2), 1217-1222 (2002)    -   PUBMED 11700324-   REFERENCE 11 (bases 1 to 4276)    -   AUTHORS Becker, K., Beales, P. L., Calver, D. M., Matthijs, G.        and Mohammed, S. N.    -   TITLE Okihiro syndrome and acro-renal-ocular syndrome: clinical        overlap, expansion of the phenotype, and absence of PAX2        mutations in two new families    -   JOURNAL J. Med. Genet. 39 (1), 68-71 (2002)    -   PUBMED 11826030    -   REMARK GeneRIF: The absence of PAX2 mutations has been        identified in two families with histories of clinical overlap of        Okihiro and acro-renal-ocular syndromes.-   REFERENCE 12 (bases 1 to 4276)    -   AUTHORS Eccles, M. R., He, S., Legge, M., Kumar, R., Fox, J.,        Zhou, C., French, M. and Tsai, R. W.    -   TITLE PAX genes in development and disease: the role of PAX2 in        urogenital tract development    -   JOURNAL Int. J. Dev. Biol. 46 (4), 535-544 (2002)    -   PUBMED 12141441    -   REMARK Review article GeneRIF: PAX2 has a role in urogenital        tract development and disease [review]-   REFERENCE 13 (bases 1 to 4276)    -   AUTHORS Chung, G. W., Edwards, A. O., Schimmenti, L. A.,        Manligas, G. S., Zhang, Y. H. and Ritter, R. III.    -   TITLE Renal-coloboma syndrome: report of a novel PAX2 gene        mutation    -   JOURNAL Am. J. Ophthalmol. 132 (6), 910-914 (2001)    -   PUBMED 11730657    -   REMARK GeneRIF: The causal relationship between PAX2 gene        mutations and renal-coloboma syndrome is further supported-   REFERENCE 14 (bases 1 to 4276)    -   AUTHORS Nishimoto, K., Iijima, K., Shirakawa, T., Kitagawa, K.,        Satomura, K., Nakamura, H. and Yoshikawa, N.    -   TITLE PAX2 gene mutation in a family with isolated renal        hypoplasia    -   JOURNAL J. Am. Soc. Nephrol. 12 (8), 1769-1772 (2001)    -   PUBMED 11461952-   REFERENCE 15 (bases 1 to 4276)    -   AUTHORS Ritz-Laser, B., Estreicher, A., Gauthier, B. and        Philippe, J.    -   TITLE The paired homeodomain transcription factor Pax-2 is        expressed in the endocrine pancreas and transactivates the        glucagon gene promoter    -   JOURNAL J. Biol. Chem. 275 (42), 32708-32715 (2000)    -   PUBMED 10938089-   REFERENCE 16 (bases 1 to 4276)    -   AUTHORS Lechner, M. S., Levitan, I. and Dressler, G. R.    -   TITLE PTIP, a novel BRCT domain-containing protein interacts        with Pax2 and is associated with active chromatin    -   JOURNAL Nucleic Acids Res. 28 (14), 2741-2751 (2000)    -   PUBMED 10908331-   REFERENCE 17 (bases 1 to 4276)    -   AUTHORS Tavassoli, K., Ruger, W. and Horst, J.    -   TITLE Alternative splicing in PAX2 generates a new reading frame        and an extended conserved coding region at the carboxy terminus    -   JOURNAL Hum. Genet. 101 (3), 371-375 (1997)    -   PUBMED 9439670-   REFERENCE 18 (bases 1 to 4276)    -   AUTHORS Dahl, E., Koseki, H. and Balling, R.    -   TITLE Pax genes and organogenesis    -   JOURNAL Bioessays 19 (9), 755-765 (1997)    -   PUBMED 9297966    -   REMARK Review article-   REFERENCE 19 (bases 1 to 4276)    -   AUTHORS Schimmenti, L. A., Cunliffe, H. E., McNoe, L. A.,        Ward, T. A., French, M. C., Shim, H. H., Zhang, Y. H.,        Proesmans, W., Leys, A., Byerly, K. A., Braddock, S. R., Masuno,        M., Imaizumi, K., Devriendt, K. and Eccles, M. R.    -   TITLE Further delineation of renal-coloboma syndrome in patients        with extreme variability of phenotype and identical PAX2        mutations    -   JOURNAL Am. J. Hum. Genet. 60 (4), 869-878 (1997)    -   PUBMED 9106533-   REFERENCE 20 (bases 1 to 4276)    -   AUTHORS Narahara, K., Baker, E., Ito, S., Yokoyama, Y., Yu, S.,        Hewitt, D., Sutherland, G. R., Eccles, M. R. and Richards, R. I.    -   TITLE Localisation of a 10q breakpoint within the PAX2 gene in a        patient with a de novo t(10;13) translocation and optic nerve        coloboma-renal disease    -   JOURNAL J. Med. Genet. 34 (3), 213-216 (1997)    -   PUBMED 9132492-   REFERENCE 21 (bases 1 to 4276)    -   AUTHORS Dehbi, M., Ghahremani, M., Lechner, M., Dressler, G. and        Pelletier, J.    -   TITLE The paired-box transcription factor, PAX2, positively        modulates expression of the Wilms' tumor suppressor gene (WT1)    -   JOURNAL Oncogene 13 (3), 447-453 (1996)    -   PUBMED 8760285-   REFERENCE 22 (bases 1 to 4276)    -   AUTHORS Sanyanusin, P., Norrish, J. H., Ward, T. A., Nebel, A.,        McNoe, L. A. and Eccles, M. R.    -   TITLE Genomic structure of the human PAX2 gene    -   JOURNAL Genomics 35 (1), 258-261 (1996)    -   PUBMED 8661132-   REFERENCE 23 (bases 1 to 4276)    -   AUTHORS Sanyanusin, P., Schimmenti, L. A., McNoe, L. A.,        Ward, T. A., Pierpont, M. E., Sullivan, M. J., Dobyns, W. B. and        Eccles, M. R.    -   TITLE Mutation of the PAX2 gene in a family with optic nerve        colobomas, renal anomalies and vesicoureteral reflux    -   JOURNAL Nat. Genet. 9 (4), 358-364 (1995)    -   PUBMED 7795640-   REFERENCE 24 (bases 1 to 4276)    -   AUTHORS Ward, T. A., Nebel, A., Reeve, A. E. and Eccles, M. R.    -   TITLE Alternative messenger RNA forms and open reading frames        within an additional conserved region of the human PAX-2 gene    -   JOURNAL Cell Growth Differ. 5 (9), 1015-1021 (1994)    -   PUBMED 7819127-   REFERENCE 25 (bases 1 to 4276)    -   AUTHORS Noll, M.    -   TITLE Evolution and role of Pax genes    -   JOURNAL Curr. Opin. Genet. Dev. 3 (4), 595-605 (1993)    -   PUBMED 8241771    -   REMARK Review article-   REFERENCE 26 (bases 1 to 4276)    -   AUTHORS Stapleton, P., Weith, A., Urbanek, P., Kozmik, Z. and        Busslinger, M.    -   TITLE Chromosomal localization of seven PAX genes and cloning of        a novel family member, PAX-9    -   JOURNAL Nat. Genet. 3 (4), 292-298 (1993)    -   PUBMED 7981748-   REFERENCE 27 (bases 1 to 4276)    -   AUTHORS Pilz, A. J., Povey, S., Gruss, P. and Abbott, C. M.    -   TITLE Mapping of the human homologs of the murine        paired-box-containing genes    -   JOURNAL Mamm. Genome 4 (2), 78-82 (1993)    -   PUBMED 8431641-   REFERENCE 28 (bases 1 to 4276)    -   AUTHORS Eccles, M. R., Wallis, L. J., Fidler, A. E., Spurr, N.        K., Goodfellow, P. J. and Reeve, A. E.    -   TITLE Expression of the PAX2 gene in human fetal kidney and        Wilms' tumor    -   JOURNAL Cell Growth Differ. 3 (5), 279-289 (1992)    -   PUBMED 1378753-   COMMENT REVIEWED REFSEQ: This record has been curated by NCBI staff.    The reference sequence was derived from U45255.1 and BM671839.1. On    Sep. 22, 2003 this sequence version replaced gi:4557822.    -   Summary: PAX2 encodes paired box gene 2, one of many human        homologues of the Drosophila melanogaster gene prd. The central        feature of this transcription factor gene family is the        conserved DNA-binding paired box domain. PAX2 is believed to be        a target of transcriptional supression by the tumor supressor        gene WT1. Mutations within PAX2 have been shown to result in        optic nerve colobomas and renal hypoplasia. Alternative splicing        of this gene results in multiple transcript variants.    -   Transcript Variant: This variant (a) uses an alternate in-frame        splice site in the 3′ coding region, compared to variant e,        resulting in a shorter protein (isoform a) that has a shorter,        distinct C-terminus compared to isoform e.    -   COMPLETENESS: complete on the 3′ end.

FEATURES Location/Qualifiers source 1 . . . 4276 /organism = “ Homosapiens” /mol_type = “ mRNA” /db_xref = “ taxon:9606” /chromosome = “10” /map = “ 10q24” gene 1 . . . 4276 /gene = “ PAX2” /db_xref = “GeneID:5076” /db_xref = “ HGNC:8616” /db_xref = “ MIM:167409” CDS 687 .. . 1937 /gene = “ PAX2” /note = “ PAX2 gene is a member of thepaired-box containing genes which encode transcription factors involvedin embryonic and fetal development; the gene product is nuclear proteinwhich binds DNA isoform a is encoded by transcript variant a; paired boxhomeotic gene 2; go_component: nucleus [goid 0005634] [evidence IEA];go_function: ATP binding [goid 0005524] [evidence IEA]; go_function: DNAbinding [goid 0003677] [evidence IEA]; go_function: DNA binding [goid0003677] [evidence TAS] [pmid 9106533]; go_function: nucleosidediphosphate kinase activity [goid 0004550] [evidence IEA]; go_process:development [goid 0007275] [evidence IEA]; go_process: transcription[goid 0006350] [evidence IEA]; go_process: CTP biosynthesis [goid0006241] [evidence IEA]; go_process: GTP biosynthesis [goid 0006183][evidence IEA]; go_process: UTP biosynthesis [goid 0006228] [evidenceIEA]; go_process: axonogenesis [goid 0007409] [evidence TAS] [pmid9106533]; go_process: cell differentiation [goid 0030154] [evidenceIEA]; go_process: visual perception [goid 0007601] [evidence TAS] [pmid9106533]; go_process: regulation of transcription, DNA-dependent [goid0006355] [evidence IEA]; go_process: transcription from RNA polymeraseII promoter [goid 0006366] [evidence TAS] [pmid 8760285]” /codon_start =1 /product = “ paired box protein 2 isoform a” /protein_id = “NP_003978.2” /db_xref = “ GI:34878699” /db_xref = “ GeneID:5076”/db_xref = “ HGNC:8616” /db_xref = “ MIM:167409” /translation = “MDMHCKADPFSAMHPGHGGVNQLGGVFVNGRPLPDVVRQRIVELAHQGVRPCDISRQLRVSHGCVSKILGRYYETGSIKPGVIGGSKPKVATPKVVDKIAEYKRQNPTMFAWEIRDRLLAEGICDNDTVPSVSSINRIIRTKVQQPFHPTPDGAGTGVTAPGHTIVPSTASPPVSSASNDPVGSYSINGILGIPRSNGEKRKRDEVEVYTDPAHIRGGGGLHLVWTLRDVSEGSVPNGDSQSGVDSLRKHLRADTFTQQQLEALDRVFERPSYPDVFQASEHIKSEQGNEYSLPALTPGLDEVKSSLSASTNPELGSNVSGTQTYPVVTGRDMASTTLPGYPPHVPPTGQGSYPTSTLAGMVPGSEFSGNPYSHPQYTAYNEAWRFS NPALL (SEQ ID NO: 41)SSPYYYSAAPRSAPAAAAAAYDRH” STS 1926 . . . 2132 /gene = “ PAX2”/standard_name = “ RH80285” /db_xref = “ UniSTS:88437” STS 3124 . . .3276 /gene = “ PAX2” /standard_name = “ D10S2478” /db_xref = “UniSTS:74159” polyA_signal 4230 . . . 4235 /gene = “ PAX2” polyA_signal4234 . . . 4239 /gene = “ PAX2” polyA_signal 4241 . . . 4246 /gene = “PAX2” polyA_site 4259 /gene = “ PAX2”

-   ORIGIN

(SEQ ID NO: 42)    1 aggctccagt ctccggccga gtcttctcgc agccgcaacccacctggggc cagcccagag   61 ctgccagcgc cgctcggctc cctccctccc tcccggcccttcggccgcgg cggcgtgcgc  121 ctgccttttc cgggggcggg ggcctggccc gcgcgctcccctcccgcagg cgccacctcg  181 gacatccccg ggattgctac ttctctgcca acttcgccaactcgccagca cttggagagg  241 cccggctccc ctcccggcgc cctctgaccg cccccgccccgcgcgctctc cgaccaccgc  301 ctctcggatg accaggttcc aggggagctg agcgagtcgcctcccccgcc cagcttcagc  361 cctggctgca gctgcagcgc gagccatgcg cccccagtgcaccccggccc ggcccaccgc  421 cccggggcca ttctgctgac cgcccagccc cgagccccgacagtggcaag ttgcggctac  481 tgcagttgca agctccggcc aacccggagg agccccagcggggagcgcag tgttgcgccc  541 cccgcccccg cgcgccccgc agcagccggg cgttcactcatcctccctcc cccaccgtcc  601 ctcccttttc tcctcaagtc ctgaagttga gtttgagaggcgacacggcg gcggcggccg  661 cgctgctccc gctcctctgc ctccccatgg atatgcactgcaaagcagac cccttctccg  721 cgatgcaccc agggcacggg ggtgtgaacc agctcgggggggtgtttgtg aacggccggc  781 ccctacccga cgtggtgagg cagcgcatcg tggagctggcccaccagggt gtgcggccct  841 gtgacatctc ccggcagctg cgggtcagcc acggctgtgtcagcaaaatc ctgggcaggt  901 actacgagac cggcagcatc aagccgggtg tgatcggtggctccaagccc aaagtggcga  961 cgcccaaagt ggtggacaag attgctgaat acaaacgacagaacccgact atgttcgcct 1021 gggagattcg agaccggctc ctggccgagg gcatctgtgacaatgacaca gtgcccagcg 1081 tctcttccat caacagaatc atccggacca aagttcagcagcctttccac ccaacgccgg 1141 atggggctgg gacaggagtg accgcccctg gccacaccattgttcccagc acggcctccc 1201 ctcctgtttc cagcgcctcc aatgacccag tgggatcctactccatcaat gggatcctgg 1261 ggattcctcg ctccaatggt gagaagagga aacgtgatgaagttgaggta tacactgatc 1321 ctgcccacat tagaggaggt ggaggtttgc atctggtctggactttaaga gatgtgtctg 1381 agggctcagt ccccaatgga gattcccaga gtggtgtggacagtttgcgg aagcacttgc 1441 gagctgacac cttcacccag cagcagctgg aagctttggatcgggtcttt gagcgtcctt 1501 cctaccctga cgtcttccag gcatcagagc acatcaaatcagaacagggg aacgagtact 1561 ccctcccagc cctgacccct gggcttgatg aagtcaagtcgagtctatct gcatccacca 1621 accctgagct gggcagcaac gtgtcaggca cacagacatacccagttgtg actggtcgtg 1681 acatggcgag caccactctg cctggttacc cccctcacgtgccccccact ggccagggaa 1741 gctaccccac ctccaccctg gcaggaatgg tgcctgggagcgagttctcc ggcaacccgt 1801 acagccaccc ccagtacacg gcctacaacg aggcttggagattcagcaac cccgccttac 1861 taagttcccc ttattattat agtgccgccc cccggtccgcccctgccgct gctgccgctg 1921 cctatgaccg ccactagtta ccgcggggac cacatcaagcttcaggccga cagcttcggc 1981 ctccacatcg tccccgtctg accccacccc ggagggagggaggaccgacg cgacgcgatg 2041 cctcccggcc accgccccag cctcacccca tcccacgacccccgcaaccc ttcacatcac 2101 ccccctcgaa ggtcggacag gacgggtgga gccgtgggcgggaccctcag gcccgggccc 2161 gccgccccca gccccgcctg ccgcccctcc ccgcctgcctggactgcgcg gcgccgtgag 2221 ggggattcgg cccagctcgt cccggcctcc accaagccagccccgaagcc cgccagccac 2281 cctgccggac tcgggcgcga cctgctggcg cgcgccggatgtttctgtga cacacaatca 2341 gcgcggaccg cagcgcggcc cagccccggg cacccgcctcggacgctcgg gcgccaggag 2401 gcttcgctgg aggggctggg ccaaggagat taagaagaaaacgactttct gcaggaggaa 2461 gagcccgctg ccgaatccct gggaaaaatt cttttcccccagtgccagcc ggactgccct 2521 cgccttccgg gtgtgccctg tcccagaaga tggaatgggggtgtgggggt ccggctctag 2581 gaacgggctt tgggggcgtc aggtctttcc aaggttgggacccaaggatc ggggggccca 2641 gcagcccgca ccgatcgagc cggactctcg gctcttcactgctcctcctg gcctgcctag 2701 ttccccaggg cccggcacct cctgctgcga gacccggctctcagccctgc cttgccccta 2761 cctcagcgtc tcttccacct gctggcctcc cagtttcccctcctgccagt ccttcgcctg 2821 tcccttgacg ccctgcatcc tcctccctga ctcgcagccccatcggacgc tctcccggga 2881 ccgccgcagg accagtttcc atagactgcg gactggggtcttcctccagc agttacttga 2941 tgccccctcc cccgacacag actctcaatc tgccggtggtaagaaccggt tctgagctgg 3001 cgtctgagct gctgcggggt ggaagtgggg ggctgcccactccactcctc ccatcccctc 3061 ccagcctcct cctccggcag gaactgaaca gaaccacaaaaagtctacat ttatttaata 3121 tgatggtctt tgcaaaaagg aacaaaacaa cacaaaagcccaccaggctg ctgctttgtg 3181 gaaagacggt gtgtgtcgtg tgaaggcgaa acccggtgtacataacccct ccccctccgc 3241 cccgccccgc ccggccccgt agagtccctg tcgcccgccggccctgcctg tagatacgcc 3301 ccgctgtctg tgctgtgaga gtcgccgctc gctgggggggaaggggggga cacagctaca 3361 cgcccattaa agcacagcac gtcctggggg aggggggcattttttatgtt acaaaaaaaa 3421 attacgaaag aaaagaaatc tctatgcaaa atgacgaacatggtcctgtg gactcctctg 3481 gcctgttttg ttggctcttt ctctgtaatt ccgtgttttcgctttttcct ccctgcccct 3541 ctctccctct gcccctctct cctctccgct tctctccccctctgtctctg tctctctccg 3601 tctctgtcgc tcttgtctgt ctgtctctgc tctttcctcggcctctctcc ccagacctgg 3661 cccggccgcc ctgtctccgc aggctagatc cgaggtggcagctccagccc ccgggctcgc 3721 cccctcgcgg gcgtgccccg cgcgccccgg gcggccgaaggccgggccgc cccgtcccgc 3781 cccgtagttg ctctttcggt agtggcgatg cgccctgcatgtctcctcac ccgtggatcg 3841 tgacgactcg aaataacaga aacaaagtca ataaagtgaaaataaataaa aatccttgaa 3901 caaatccgaa aaggcttgga gtcctcgccc agatctctctcccctgcgag ccctttttat 3961 ttgagaagga aaaagagaaa agagaatcgt ttaagggaacccggcgccca gccaggctcc 4021 agtggcccga acggggcggc gagggcggcg agggcgccgaggtccggccc atcccagtcc 4081 tgtggggctg gccgggcaga gaccccggac ccaggcccaggcctaacctg ctaaatgtcc 4141 ccggacggtt ctggtctcct cggccacttt cagtgcgtcggttcgttttg attctttttc 4201 ttttgtgcac ataagaaata aataataata ataaataaagaataaaattt tgtatgtcaa 4261 aaaaaaaaaa aaaaaa

Appendix C

-   LOCUS NM_(—)000278 4207 by mRNA linear PRI 24 Sep. 2005-   DEFINITION Homo sapiens paired box gene 2 (PAX2), transcript variant    b, mRNA.-   ACCESSION NM_(—)000278-   VERSION NM_(—)000278.2 GI:34878700-   KEYWORDS .-   SOURCE Homo sapiens (human)    -   ORGANISM Homo sapiens Eukaryota; Metazoa; Chordata; Craniata;        Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires;        Primates; Catarrhini; Hominidae; Homo.-   REFERENCE 1 (bases 1 to 4207)    -   AUTHORS Yoshimura, K., Yoshida, S., Yamaji, Y., Komori, A.,        Yoshida, A., Hatae, K., Kubota, T. and Ishibashi, T.    -   TITLE De novo insG619 mutation in PAX2 gene in a Japanese        patient with papillorenal syndrome    -   JOURNAL Am. J. Ophthalmol. 139 (4), 733-735 (2005)    -   PUBMED 15808183    -   REMARK GeneRIF: Molecular genetic analysis of the PAX2 gene in        combination with renal ultrasonography can help in making an        earlier diagnosis of the disease.-   REFERENCE 2 (bases 1 to 4207)    -   AUTHORS Mazal, P. R., Stichenwirth, M., Koller, A., Blach, S.,        Haitel, A. and Susani, M.    -   TITLE Expression of aquaporins and PAX-2 compared to CD10 and        cytokeratin 7 in renal neoplasms: a tissue microarray study    -   JOURNAL Mod. Pathol. 18 (4), 535-540 (2005)    -   PUBMED 15502805    -   REMARK GeneRIF: PAX-2 is a reliable marker for clear cell renal        cell carcinomas of lower grades but not for higher grades.-   REFERENCE 3 (bases 1 to 4207)    -   AUTHORS Higashide, T., Wada, T., Sakurai, M., Yokoyama, H. and        Sugiyama, K.    -   TITLE Macular abnormalities and optic disk anomaly associated        with a new PAX2 missense mutation    -   JOURNAL Am. J. Ophthalmol. 139 (1), 203-205 (2005)    -   PUBMED 15652857    -   REMARK GeneRIF: A new PAX2 missense mutation, R71T, may cause        macular abnormalities in addition to anomalies of the optic disk        and the kidney.-   REFERENCE 4 (bases 1 to 4207)    -   AUTHORS Buttiglieri, S., Deregibus, M. C., Bravo, S., Cassoni,        P., Chiarle, R., Bussolati, B. and Camussi, G.    -   TITLE Role of Pax2 in apoptosis resistance and proinvasive        phenotype of Kaposi's sarcoma cells    -   JOURNAL J. Biol. Chem. 279 (6), 4136-4143 (2004)    -   PUBMED 14627715    -   REMARK GeneRIF: expression of Pax2 by Kaposi's sarcoma cells        correlated with an enhanced resistance against apoptotic signals        and with the proinvasive phenotype-   REFERENCE 5 (bases 1 to 4207)    -   AUTHORS Brophy, P. D., Lang, K. M. and Dressler, G. R.    -   TITLE The secreted frizzled related protein 2 (SFRP2) gene is a        target of the Pax2 transcription factor    -   JOURNAL J. Biol. Chem. 278 (52), 52401-52405 (2003)    -   PUBMED 14561758    -   REMARK GeneRIF: Pax2 protein regulates expression of secreted        frizzled related protein 2-   REFERENCE 6. (bases 1 to 4207)    -   AUTHORS Schimmenti, L. A., Manligas, G. S. and Sieving, P. A.    -   TITLE Optic nerve dysplasia and renal insufficiency in a family        with a novel PAX2 mutation, Arg115X: further ophthalmologic        delineation of the renal-coloboma syndrome    -   JOURNAL Ophthalmic Genet. 24 (4), 191-202 (2003)    -   PUBMED 14566649    -   REMARK GeneRIF: PAX2 mutation is associated with Optic nerve        dysplasia and renal insufficiency of the renal-coloboma syndrome-   REFERENCE 7 (bases 1 to 4207)    -   AUTHORS Muratovska, A., Zhou, C., He, S., Goodyer, P. and        Eccles, M. R.    -   TITLE Paired-Box genes are frequently expressed in cancer and        often required for cancer cell survival    -   JOURNAL Oncogene 22 (39), 7989-7997 (2003)    -   PUBMED 12970747    -   REMARK GeneRIF: The PAX2 gene was frequently expressed in a        panel of 406 common primary tumor tissues and endogenous PAX        gene expression is often required for the growth and survival of        cancer cells-   REFERENCE 8 (bases 1 to 4207)    -   AUTHORS Gough, S. M., McDonald, M., Chen, X. N., Korenberg, J.        R., Neri, A., Kahn, T., Eccles, M. R. and Morris, C. M.    -   TITLE Refined physical map of the human PAX2/HOX11/NFKB2 cancer        gene region at 10q24 and relocalization of the HPV6AI1 viral        integration site to 14q13.3-q21.1    -   JOURNAL BMC Genomics 4 (1), 9 (2003)    -   PUBMED 12697057-   REFERENCE 9 (bases 1 to 4207)    -   AUTHORS Hoffmeister, A., Ropolo, A., Vasseur, S., Mallo, G. V.,        Bodeker, H., Ritz-Laser, B., Dressler, G. R., Vaccaro, M. I.,        Dagorn, J. C., Moreno, S. and Iovanna, J. L.    -   TITLE The HMG-I/Y-related protein p8 binds to p300 and Pax2        trans-activation domain-interacting protein to regulate the        trans-activation activity of the Pax2A and Pax2B transcription        factors on the glucagon gene promoter    -   JOURNAL J. Biol. Chem. 277 (25), 22314-22319 (2002)    -   PUBMED 11940591    -   REMARK GeneRIF: The HMG-I/Y-related protein p8 binds to p300 and        Pax2 trans-activation domain-interacting protein to regulate the        trans-activation activity of the Pax2A and Pax2B transcription        factors on the glucagon gene promoter.-   REFERENCE 10 (bases 1 to 4207)    -   AUTHORS Cai, Y., Lechner, M. S., Nihalani, D., Prindle, M. J.,        Holzman, L. B. and Dressler, G. R.    -   TITLE Phosphorylation of Pax2 by the c-Jun N-terminal kinase and        enhanced Pax2-dependent transcription activation    -   JOURNAL J. Biol. Chem. 277 (2), 1217-1222 (2002)    -   PUBMED 11700324-   REFERENCE 11 (bases 1 to 4207)    -   AUTHORS Becker, K., Beales, P. L., Calver, D. M., Matthijs, G.        and Mohammed, S. N.    -   TITLE Okihiro syndrome and acro-renal-ocular syndrome: clinical        overlap, expansion of the phenotype, and absence of PAX2        mutations in two new families    -   JOURNAL J. Med. Genet. 39 (1), 68-71 (2002)    -   PUBMED 11826030    -   REMARK GeneRIF: The absence of PAX2 mutations has been        identified in two families with histories of clinical overlap of        Okihiro and acro-renal-ocular syndromes.-   REFERENCE 12 (bases 1 to 4207)    -   AUTHORS Eccles, M. R., He, S., Legge, M., Kumar, R., Fox, J.,        Zhou, C., French, M. and Tsai, R. W.    -   TITLE PAX genes in development and disease: the role of PAX2 in        urogenital tract development    -   JOURNAL Int. J. Dev. Biol. 46 (4), 535-544 (2002)    -   PUBMED 12141441    -   REMARK Review article GeneRIF: PAX2 has a role in urogenital        tract development and disease [review]-   REFERENCE 13 (bases 1 to 4207)    -   AUTHORS Chung, G. W., Edwards, A. O., Schimmenti, L. A.,        Manligas, G. S., Zhang, Y. H. and Ritter, R. III.    -   TITLE Renal-coloboma syndrome: report of a novel PAX2 gene        mutation    -   JOURNAL Am. J. Ophthalmol. 132 (6), 910-914 (2001)    -   PUBMED 11730657    -   REMARK GeneRIF: The causal relationship between PAX2 gene        mutations and renal-coloboma syndrome is further supported-   REFERENCE 14 (bases 1 to 4207)    -   AUTHORS Nishimoto, K., Iijima, K., Shirakawa, T., Kitagawa, K.,        Satomura, K., Nakamura, H. and Yoshikawa, N.    -   TITLE PAX2 gene mutation in a family with isolated renal        hypoplasia    -   JOURNAL J. Am. Soc. Nephrol. 12 (8), 1769-1772 (2001)    -   PUBMED 11461952-   REFERENCE 15 (bases 1 to 4207)    -   AUTHORS Ritz-Laser, B., Estreicher, A., Gauthier, B. and        Philippe, J.    -   TITLE The paired homeodomain transcription factor Pax-2 is        expressed in the endocrine pancreas and transactivates the        glucagon gene promoter    -   JOURNAL J. Biol. Chem. 275 (42), 32708-32715 (2000)    -   PUBMED 10938089-   REFERENCE 16 (bases 1 to 4207)    -   AUTHORS Lechner, M. S., Levitan, I. and Dressler, G. R.    -   TITLE PTIP, a novel BRCT domain-containing protein interacts        with Pax2 and is associated with active chromatin    -   JOURNAL Nucleic Acids Res. 28 (14), 2741-2751 (2000)    -   PUBMED 10908331-   REFERENCE 17 (bases 1 to 4207)    -   AUTHORS Tavassoli, K., Ruger, W. and Horst, J.    -   TITLE Alternative splicing in PAX2 generates a new reading frame        and an extended conserved coding region at the carboxy terminus    -   JOURNAL Hum. Genet. 101 (3), 371-375 (1997)    -   PUBMED 9439670-   REFERENCE 18 (bases 1 to 4207)    -   AUTHORS Dahl, E., Koseki, H. and Balling, R.    -   TITLE Pax genes and organogenesis    -   JOURNAL Bioessays 19 (9), 755-765 (1997)    -   PUBMED 9297966    -   REMARK Review article-   REFERENCE 19 (bases 1 to 4207)    -   AUTHORS Schimmenti, L. A., Cunliffe, H. E., McNoe, L. A.,        Ward, T. A., French, M. C., Shim, H. H., Zhang, Y. H.,        Proesmans, W., Leys, A., Byerly, K. A., Braddock, S. R., Masuno,        M., Imaizumi, K., Devriendt, K. and Eccles, M. R.    -   TITLE Further delineation of renal-coloboma syndrome in patients        with extreme variability of phenotype and identical PAX2        mutations    -   JOURNAL Am. J. Hum. Genet. 60 (4), 869-878 (1997)    -   PUBMED 9106533-   REFERENCE 20 (bases 1 to 4207)    -   AUTHORS Narahara, K., Baker, E., Ito, S., Yokoyama, Y., Yu, S.,        Hewitt, D., Sutherland, G. R., Eccles, M. R. and Richards, R. I.    -   TITLE Localisation of a 10q breakpoint within the PAX2 gene in a        patient with a de novo t(10;13) translocation and optic nerve        coloboma-renal disease    -   JOURNAL J. Med. Genet. 34 (3), 213-216 (1997)    -   PUBMED 9132492-   REFERENCE 21 (bases 1 to 4207)    -   AUTHORS Dehbi, M., Ghahremani, M., Lechner, M., Dressler, G. and        Pelletier, J.    -   TITLE The paired-box transcription factor, PAX2, positively        modulates expression of the Wilms' tumor suppressor gene (WT1)    -   JOURNAL Oncogene 13 (3), 447-453 (1996)    -   PUBMED 8760285-   REFERENCE 22 (bases 1 to 4207)    -   AUTHORS Sanyanusin, P., Norrish, J. H., Ward, T. A., Nebel, A.,        McNoe, L. A. and Eccles, M. R.    -   TITLE Genomic structure of the human PAX2 gene    -   JOURNAL Genomics 35 (1), 258-261 (1996)    -   PUBMED 8661132-   REFERENCE 23 (bases 1 to 4207)    -   AUTHORS Sanyanusin, P., Schimmenti, L. A., McNoe, L. A.,        Ward, T. A., Pierpont, M. E., Sullivan, M. J., Dobyns, W. B. and        Eccles, M. R.    -   TITLE Mutation of the PAX2 gene in a family with optic nerve        colobomas, renal anomalies and vesicoureteral reflux    -   JOURNAL Nat. Genet. 9 (4), 358-364 (1995)    -   PUBMED 7795640-   REFERENCE 24 (bases 1 to 4207)    -   AUTHORS Ward, T. A., Nebel, A., Reeve, A. E. and Eccles, M. R.    -   TITLE Alternative messenger RNA forms and open reading frames        within an additional conserved region of the human PAX-2 gene    -   JOURNAL Cell Growth Differ. 5 (9), 1015-1021 (1994)    -   PUBMED 7819127-   REFERENCE 25 (bases 1 to 4207)    -   AUTHORS Noll, M.    -   TITLE Evolution and role of Pax genes    -   JOURNAL Curr. Opin. Genet. Dev. 3 (4), 595-605 (1993)    -   PUBMED 8241771    -   REMARK Review article-   REFERENCE 26 (bases 1 to 4207)    -   AUTHORS Stapleton, P., Weith, A., Urbanek, P., Kozmik, Z. and        Busslinger, M.    -   TITLE Chromosomal localization of seven PAX genes and cloning of        a novel family member, PAX-9    -   JOURNAL Nat. Genet. 3 (4), 292-298 (1993)    -   PUBMED 7981748-   REFERENCE 27 (bases 1 to 4207)    -   AUTHORS Pilz, A. J., Povey, S., Gruss, P. and Abbott, C. M.    -   TITLE Mapping of the human homologs of the murine        paired-box-containing genes    -   JOURNAL Mamm. Genome 4 (2), 78-82 (1993)    -   PUBMED 8431641-   REFERENCE 28 (bases 1 to 4207)    -   AUTHORS Eccles, M. R., Wallis, L. J., Fidler, A. E., Spurr, N.        K., Goodfellow, P. J. and Reeve, A. E.    -   TITLE Expression of the PAX2 gene in human fetal kidney and        Wilms' tumor    -   JOURNAL Cell Growth Differ. 3 (5), 279-289 (1992)    -   PUBMED 1378753-   COMMENT REVIEWED REFSEQ: This record has been curated by NCBI staff.    The reference sequence was derived from U45255.1 and BM671839.1. On    Sep. 22, 2003 this sequence version replaced gi:4557820.    -   Summary: PAX2 encodes paired box gene 2, one of many human        homologues of the Drosophila melanogaster gene prd. The central        feature of this transcription factor gene family is the        conserved DNA-binding paired box domain. PAX2 is believed to be        a target of transcriptional supression by the tumor supressor        gene WT1. Mutations within PAX2 have been shown to result in        optic nerve colobomas and renal hypoplasia. Alternative splicing        of this gene results in multiple transcript variants.    -   Transcript Variant: This variant (b) lacks an alternate in-frame        exon and uses an alternate splice site in the 3′ coding region,        compared to variant e. This results in a protein (isoform b)        with a shorter, distinct C-terminus compared to isoform e.    -   COMPLETENESS: complete on the 3′ end.

FEATURES Location/Qualifiers source 1 . . . 4207 /organism = “ Homosapiens” /mol_type = “ mRNA” /db_xref = “ taxon:9606” /chromosome = “10” /map = “ 10q24” gene 1 . . . 4207 /gene = “ PAX2” /db_xref = “GeneID:5076” /db_xref = “ HGNC:8616” /db_xref = “ MIM:167409” CDS 687 .. . 1868 /gene = “ PAX2” /note = “ PAX2 gene is a member of thepaired-box containing genes which encode transcription factors involvedin embryonic and fetal development; the gene product is nuclear proteinwhich binds DNA isoform b is encoded by transcript variant b; paired boxhomeotic gene 2; go_component: nucleus [goid 0005634] [evidence IEA];go_function: ATP binding [goid 0005524] [evidence IEA]; go_function: DNAbinding [goid 0003677] [evidence IEA]; go_function: DNA binding [goid0003677] [evidence TAS] [pmid 9106533]; go_function: nucleosidediphosphate kinase activity [goid 0004550] [evidence IEA]; go_process:development [goid 0007275] [evidence IEA]; go_process: transcription[goid 0006350] [evidence IEA]; go_process: CTP biosynthesis [goid0006241] [evidence IEA]; go_process: GTP biosynthesis [goid 0006183][evidence IEA]; go_process: UTP biosynthesis [goid 0006228] [evidenceIEA]; go_process: axonogenesis [goid 0007409] [evidence TAS] [pmid9106533]; go_process: cell differentiation [goid 0030154] [evidenceIEA]; go_process: visual perception [goid 0007601] [evidence TAS] [pmid9106533]; go_process: regulation of transcription, DNA-dependent [goid0006355] [evidence IEA]; go_process: transcription from RNA polymeraseII promoter [goid 0006366] [evidence TAS] [pmid 8760285]” /codon_start =1 /product = “ paired box protein 2 isoform b” /protein_id = “NP_000269.2” /db_xref = “ GI:34878701” /db_xref = “ GeneID:5076”/db_xref = “ HGNC:8616” /db_xref = “ MIM:167409” /translation = “MDMHCKADPFSAMHPGHGGVNQLGGVFVNGRPLPDVVRQRIVELAHQGVRPCDISRQLRVSHGCVSKILGRYYETGSIKPGVIGGSKPKVATPKVVDKIAEYKRQNPTMFAWEIRDRLLAEGICDNDTVPSVSSINRIIRTKVQQPFHPTPDGAGTGVTAPGHTIVPSTASPPVSSASNDPVGSYSINGILGIPRSNGEKRKRDEDVSEGSVPNGDSQSGVDSLRKHLRADTFTQQQLEALDRVFERPSYPDVFQASEHIKSEQGNEYSLPALTPGLDEVKSSLSASTNPELGSNVSGTQTYPVVTGRDMASTTLPGYPPHVPPTGQGSYPTSTLAGMVPGSEFSGNPYSHPQYTAYNEAWRFSNPALLSSPYYYSAAPRSAPAAAAAAYDR (SEQ ID NO: 43) H” STS 1857 . . .2063 /gene = “ PAX2” /standard_name = “ RH80285” /db_xref = “UniSTS:88437” STS 3055 . . . 3207 /gene = “ PAX2” /standard_name = “D10S2478” /db_xref = “ UniSTS:74159” polyA_signal 4161 . . . 4166 /gene= “ PAX2” polyA_signal 4165 . . . 4170 /gene = “ PAX2” polyA_signal 4172. . . 4177 /gene = “ PAX2” polyA_site 4190 /gene = “ PAX2”

-   ORIGIN

(SEQ ID NO: 44)    1 aggctccagt ctccggccga gtcttctcgc agccgcaacccacctggggc cagcccagag   61 ctgccagcgc cgctcggctc cctccctccc tcccggcccttcggccgcgg cggcgtgcgc  121 ctgccttttc cgggggcggg ggcctggccc gcgcgctcccctcccgcagg cgccacctcg  181 gacatccccg ggattgctac ttctctgcca acttcgccaactcgccagca cttggagagg  241 cccggctccc ctcccggcgc cctctgaccg cccccgccccgcgcgctctc cgaccaccgc  301 ctctcggatg accaggttcc aggggagctg agcgagtcgcctcccccgcc cagcttcagc  361 cctggctgca gctgcagcgc gagccatgcg cccccagtgcaccccggccc ggcccaccgc  421 cccggggcca ttctgctgac cgcccagccc cgagccccgacagtggcaag ttgcggctac  481 tgcagttgca agctccggcc aacccggagg agccccagcggggagcgcag tgttgcgccc  541 cccgcccccg cgcgccccgc agcagccggg cgttcactcatcctccctcc cccaccgtcc  601 ctcccttttc tcctcaagtc ctgaagttga gtttgagaggcgacacggcg gcggcggccg  661 cgctgctccc gctcctctgc ctccccatgg atatgcactgcaaagcagac cccttctccg  721 cgatgcaccc agggcacggg ggtgtgaacc agctcgggggggtgtttgtg aacggccggc  781 ccctacccga cgtggtgagg cagcgcatcg tggagctggcccaccagggt gtgcggccct  841 gtgacatctc ccggcagctg cgggtcagcc acggctgtgtcagcaaaatc ctgggcaggt  901 actacgagac cggcagcatc aagccgggtg tgatcggtggctccaagccc aaagtggcga  961 cgcccaaagt ggtggacaag attgctgaat acaaacgacagaacccgact atgttcgcct 1021 gggagattcg agaccggctc ctggccgagg gcatctgtgacaatgacaca gtgcccagcg 1081 tctcttccat caacagaatc atccggacca aagttcagcagcctttccac ccaacgccgg 1141 atggggctgg gacaggagtg accgcccctg gccacaccattgttcccagc acggcctccc 1201 ctcctgtttc cagcgcctcc aatgacccag tgggatcctactccatcaat gggatcctgg 1261 ggattcctcg ctccaatggt gagaagagga aacgtgatgaagatgtgtct gagggctcag 1321 tccccaatgg agattcccag agtggtgtgg acagtttgcggaagcacttg cgagctgaca 1381 ccttcaccca gcagcagctg gaagctttgg atcgggtctttgagcgtcct tcctaccctg 1441 acgtcttcca ggcatcagag cacatcaaat cagaacaggggaacgagtac tccctcccag 1501 ccctgacccc tgggcttgat gaagtcaagt cgagtctatctgcatccacc aaccctgagc 1561 tgggcagcaa cgtgtcaggc acacagacat acccagttgtgactggtcgt gacatggcga 1621 gcaccactct gcctggttac ccccctcacg tgccccccactggccaggga agctacccca 1681 cctccaccct ggcaggaatg gtgcctggga gcgagttctccggcaacccg tacagccacc 1741 cccagtacac ggcctacaac gaggcttgga gattcagcaaccccgcctta ctaagttccc 1801 cttattatta tagtgccgcc ccccggtccg cccctgccgctgctgccgct gcctatgacc 1861 gccactagtt accgcgggga ccacatcaag cttcaggccgacagcttcgg cctccacatc 1921 gtccccgtct gaccccaccc cggagggagg gaggaccgacgcgacgcgat gcctcccggc 1981 caccgcccca gcctcacccc atcccacgac ccccgcaacccttcacatca cccccctcga 2041 aggtcggaca ggacgggtgg agccgtgggc gggaccctcaggcccgggcc cgccgccccc 2101 agccccgcct gccgcccctc cccgcctgcc tggactgcgcggcgccgtga gggggattcg 2161 gcccagctcg tcccggcctc caccaagcca gccccgaagcccgccagcca ccctgccgga 2221 ctcgggcgcg acctgctggc gcgcgccgga tgtttctgtgacacacaatc agcgcggacc 2281 gcagcgcggc ccagccccgg gcacccgcct cggacgctcgggcgccagga ggcttcgctg 2341 gaggggctgg gccaaggaga ttaagaagaa aacgactttctgcaggagga agagcccgct 2401 gccgaatccc tgggaaaaat tcttttcccc cagtgccagccggactgccc tcgccttccg 2461 ggtgtgccct gtcccagaag atggaatggg ggtgtgggggtccggctcta ggaacgggct 2521 ttgggggcgt caggtctttc caaggttggg acccaaggatcggggggccc agcagcccgc 2581 accgatcgag ccggactctc ggctcttcac tgctcctcctggcctgccta gttccccagg 2641 gcccggcacc tcctgctgcg agacccggct ctcagccctgccttgcccct acctcagcgt 2701 ctcttccacc tgctggcctc ccagtttccc ctcctgccagtccttcgcct gtcccttgac 2761 gccctgcatc ctcctccctg actcgcagcc ccatcggacgctctcccggg accgccgcag 2821 gaccagtttc catagactgc ggactggggt cttcctccagcagttacttg atgccccctc 2881 ccccgacaca gactctcaat ctgccggtgg taagaaccggttctgagctg gcgtctgagc 2941 tgctgcgggg tggaagtggg gggctgccca ctccactcctcccatcccct cccagcctcc 3001 tcctccggca ggaactgaac agaaccacaa aaagtctacatttatttaat atgatggtct 3061 ttgcaaaaag gaacaaaaca acacaaaagc ccaccaggctgctgctttgt ggaaagacgg 3121 tgtgtgtcgt gtgaaggcga aacccggtgt acataacccctccccctccg ccccgccccg 3181 cccggccccg tagagtccct gtcgcccgcc ggccctgcctgtagatacgc cccgctgtct 3241 gtgctgtgag agtcgccgct cgctgggggg gaagggggggacacagctac acgcccatta 3301 aagcacagca cgtcctgggg gaggggggca ttttttatgttacaaaaaaa aattacgaaa 3361 gaaaagaaat ctctatgcaa aatgacgaac atggtcctgtggactcctct ggcctgtttt 3421 gttggctctt tctctgtaat tccgtgtttt cgctttttcctccctgcccc tctctccctc 3481 tgcccctctc tcctctccgc ttctctcccc ctctgtctctgtctctctcc gtctctgtcg 3541 ctcttgtctg tctgtctctg ctctttcctc ggcctctctccccagacctg gcccggccgc 3601 cctgtctccg caggctagat ccgaggtggc agctccagcccccgggctcg ccccctcgcg 3661 ggcgtgcccc gcgcgccccg ggcggccgaa ggccgggccgccccgtcccg ccccgtagtt 3721 gctctttcgg tagtggcgat gcgccctgca tgtctcctcacccgtggatc gtgacgactc 3781 gaaataacag aaacaaagtc aataaagtga aaataaataaaaatccttga acaaatccga 3841 aaaggcttgg agtcctcgcc cagatctctc tcccctgcgagcccttttta tttgagaagg 3901 aaaaagagaa aagagaatcg tttaagggaa cccggcgcccagccaggctc cagtggcccg 3961 aacggggcgg cgagggcggc gagggcgccg aggtccggcccatcccagtc ctgtggggct 4021 ggccgggcag agaccccgga cccaggccca ggcctaacctgctaaatgtc cccggacggt 4081 tctggtctcc tcggccactt tcagtgcgtc ggttcgttttgattcttttt cttttgtgca 4141 cataagaaat aaataataat aataaataaa gaataaaattttgtatgtca aaaaaaaaaa 4201 aaaaaaa

Appendix D

-   LOCUS NM_(—)003988 4290 bp mRNA linear PRI Sep. 24, 2005-   DEFINITION Homo sapiens paired box gene 2 (PAX2), transcript variant    c, mRNA.-   ACCESSION NM_(—)003988-   VERSION NM_(—)003988.2 GI:34878708-   KEYWORDS.-   SOURCE Homo sapiens (human)    -   ORGANISM Homo sapiens Eukaryota; Metazoa; Chordata; Craniata;        Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires;        Primates; Catarrhini; Hominidae; Homo.-   REFERENCE 1 (bases 1 to 4290)    -   AUTHORS Yoshimura, K., Yoshida, S., Yamaji, Y., Komori, A.,        Yoshida, A., Hatae, K., Kubota, T. and Ishibashi, T.    -   TITLE De novo insG619 mutation in PAX2 gene in a Japanese        patient with papillorenal syndrome    -   JOURNAL Am. J. Ophthalmol. 139 (4), 733-735 (2005)    -   PUBMED 15808183    -   REMARK GeneRIF: Molecular genetic analysis of the PAX2 gene in        combination with renal ultrasonography can help in making an        earlier diagnosis of the disease.-   REFERENCE 2 (bases 1 to 4290)    -   AUTHORS Mazal, P. R., Stichenwirth, M., Koller, A., Blach, S.,        Haitel, A. and Susani, M.    -   TITLE Expression of aquaporins and PAX-2 compared to CD10 and        cytokeratin 7 in renal neoplasms: a tissue microarray study    -   JOURNAL Mod. Pathol. 18 (4), 535-540 (2005)    -   PUBMED 15502805    -   REMARK GeneRIF: PAX-2 is a reliable marker for clear cell renal        cell carcinomas of lower grades but not for higher grades.-   REFERENCE 3 (bases 1 to 4290)    -   AUTHORS Higashide, T., Wada, T., Sakurai, M., Yokoyama, H. and        Sugiyama, K.    -   TITLE Macular abnormalities and optic disk anomaly associated        with a new PAX2 missense mutation    -   JOURNAL Am. J. Ophthalmol. 139 (1), 203-205 (2005)    -   PUBMED 15652857    -   REMARK GeneRIF: A new PAX2 missense mutation, R71T, may cause        macular abnormalities in addition to anomalies of the optic disk        and the kidney.-   REFERENCE 4 (bases 1 to 4290)    -   AUTHORS Buttiglieri, S., Deregibus, M. C., Bravo, S., Cassoni,        P., Chiarle, R., Bussolati, B. and Camussi, G.    -   TITLE Role of Pax2 in apoptosis resistance and proinvasive        phenotype of Kaposi's sarcoma cells    -   JOURNAL J. Biol. Chem. 279 (6), 4136-4143 (2004)    -   PUBMED 14627715    -   REMARK GeneRIF: expression of Pax2 by Kaposi's sarcoma cells        correlated with an enhanced resistance against apoptotic signals        and with the proinvasive phenotype-   REFERENCE 5 (bases 1 to 4290)    -   AUTHORS Brophy, P. D., Lang, K. M. and Dressler, G. R.    -   TITLE The secreted frizzled related protein 2 (SFRP2) gene is a        target of the Pax2 transcription factor    -   JOURNAL J. Biol. Chem. 278 (52), 52401-52405 (2003)    -   PUBMED 14561758    -   REMARK GeneRIF: Pax2 protein regulates expression of secreted        frizzled related protein 2-   REFERENCE 6 (bases 1 to 4290)    -   AUTHORS Schimmenti, L. A., Manligas, G. S. and Sieving, P. A.    -   TITLE Optic nerve dysplasia and renal insufficiency in a family        with a novel PAX2 mutation, Arg115X: further ophthalmologic        delineation of the renal-coloboma syndrome    -   JOURNAL Ophthalmic Genet. 24 (4), 191-202 (2003)    -   PUBMED 14566649    -   REMARK GeneRIF: PAX2 mutation is associated with Optic nerve        dysplasia and renal insufficiency of the renal-coloboma syndrome-   REFERENCE 7 (bases 1 to 4290)    -   AUTHORS Muratovska, A., Zhou, C., He, S., Goodyer, P. and        Eccles, M. R.    -   TITLE Paired-Box genes are frequently expressed in cancer and        often required for cancer cell survival    -   JOURNAL Oncogene 22 (39), 7989-7997 (2003)    -   PUBMED 12970747    -   REMARK GeneRIF: The PAX2 gene was frequently expressed in a        panel of 406 common primary tumor tissues and endogenous PAX        gene expression is often required for the growth and survival of        cancer cells-   REFERENCE 8 (bases 1 to 4290)    -   AUTHORS Gough, S. M., McDonald, M., Chen, X. N., Korenberg, J.        R., Neri, A., Kahn, T., Eccles, M. R. and Morris, C. M.    -   TITLE Refined physical map of the human PAX2/HOX11/NFKB2 cancer        gene region at 10q24 and relocalization of the HPV6AI1 viral        integration site to 14q13.3-q21.1    -   JOURNAL BMC Genomics 4 (1), 9 (2003)    -   PUBMED 12697057-   REFERENCE 9 (bases 1 to 4290)    -   AUTHORS Hoffmeister, A., Ropolo, A., Vasseur, S., Mallo, G. V.,        Bodeker, H., Ritz-Laser, B., Dressler, G. R., Vaccaro, M. I.,        Dagorn, J. C., Moreno, S. and Iovanna, J. L.    -   TITLE The HMG-I/Y-related protein p8 binds to p300 and Pax2        trans-activation domain-interacting protein to regulate the        trans-activation activity of the Pax2A and Pax2B transcription        factors on the glucagon gene promoter    -   JOURNAL J. Biol. Chem. 277 (25), 22314-22319 (2002)    -   PUBMED 11940591    -   REMARK GeneRIF: The HMG-I/Y-related protein p8 binds to p300 and        Pax2 trans-activation domain-interacting protein to regulate the        trans-activation activity of the Pax2A and Pax2B transcription        factors on the glucagon gene promoter.-   REFERENCE 10 (bases 1 to 4290)    -   AUTHORS Cai, Y., Lechner, M. S., Nihalani, D., Prindle, M. J.,        Holzman, L. B. and Dressler, G. R.    -   TITLE Phosphorylation of Pax2 by the c-Jun N-terminal kinase and        enhanced Pax2-dependent transcription activation    -   JOURNAL J. Biol. Chem. 277 (2), 1217-1222 (2002)    -   PUBMED 11700324-   REFERENCE 11 (bases 1 to 4290)    -   AUTHORS Becker, K., Beales, P. L., Calver, D. M., Matthijs, G.        and Mohammed, S. N.    -   TITLE Okihiro syndrome and acro-renal-ocular syndrome: clinical        overlap, expansion of the phenotype, and absence of PAX2        mutations in two new families    -   JOURNAL J. Med. Genet. 39 (1), 68-71 (2002)    -   PUBMED 11826030    -   REMARK GeneRIF: The absence of PAX2 mutations has been        identified in two families with histories of clinical overlap of        Okihiro and acro-renal-ocular syndromes.-   REFERENCE 12 (bases 1 to 4290)    -   AUTHORS Eccles, M. R., He, S., Legge, M., Kumar, R., Fox, J.,        Zhou, C., French, M. and Tsai, R. W.    -   TITLE PAX genes in development and disease: the role of PAX2 in        urogenital tract development    -   JOURNAL Int. J. Dev. Biol. 46 (4), 535-544 (2002)    -   PUBMED 12141441    -   REMARK Review article GeneRIF: PAX2 has a role in urogenital        tract development and disease [review]-   REFERENCE 13 (bases 1 to 4290)    -   AUTHORS Chung, G. W., Edwards, A. O., Schimmenti, L. A.,        Manligas, G. S., Zhang, Y. H. and Ritter, R. III.    -   TITLE Renal-coloboma syndrome: report of a novel PAX2 gene        mutation    -   JOURNAL Am. J. Ophthalmol. 132 (6), 910-914 (2001)    -   PUBMED 11730657    -   REMARK GeneRIF: The causal relationship between PAX2 gene        mutations and renal-coloboma syndrome is further supported-   REFERENCE 14 (bases 1 to 4290)    -   AUTHORS Nishimoto, K., Iijima, K., Shirakawa, T., Kitagawa, K.,        Satomura, K., Nakamura, H. and Yoshikawa, N.    -   TITLE PAX2 gene mutation in a family with isolated renal        hypoplasia    -   JOURNAL J. Am. Soc. Nephrol. 12 (8), 1769-1772 (2001)    -   PUBMED 11461952-   REFERENCE 15 (bases 1 to 4290)    -   AUTHORS Ritz-Laser, B., Estreicher, A., Gauthier, B. and        Philippe, J.    -   TITLE The paired homeodomain transcription factor Pax-2 is        expressed in the endocrine pancreas and transactivates the        glucagon gene promoter    -   JOURNAL J. Biol. Chem. 275 (42), 32708-32715 (2000)    -   PUBMED 10938089-   REFERENCE 16 (bases 1 to 4290)    -   AUTHORS Lechner, M. S., Levitan, I. and Dressler, G. R.    -   TITLE PTIP, a novel BRCT domain-containing protein interacts        with Pax2 and is associated with active chromatin    -   JOURNAL Nucleic Acids Res. 28 (14), 2741-2751 (2000)    -   PUBMED 10908331-   REFERENCE 17 (bases 1 to 4290)    -   AUTHORS Tavassoli, K., Ruger, W. and Horst, J.    -   TITLE Alternative splicing in PAX2 generates a new reading frame        and an extended conserved coding region at the carboxy terminus    -   JOURNAL Hum. Genet. 101 (3), 371-375 (1997)    -   PUBMED 9439670-   REFERENCE 18 (bases 1 to 4290)    -   AUTHORS Dahl, E., Koseki, H. and Balling, R.    -   TITLE Pax genes and organogenesis    -   JOURNAL Bioessays 19 (9), 755-765 (1997)    -   PUBMED 9297966    -   REMARK Review article-   REFERENCE 19 (bases 1 to 4290)    -   AUTHORS Schimmenti, L. A., Cunliffe, H. E., McNoe, L. A.,        Ward, T. A., French, M. C., Shim, H. H., Zhang, Y. H.,        Proesmans, W., Leys, A., Byerly, K. A., Braddock, S. R., Masuno,        M., Imaizum i, K., Devriendt, K. and Eccles, M. R.    -   TITLE Further delineation of renal-coloboma syndrome in patients        with extreme variability of phenotype and identical PAX2        mutations    -   JOURNAL Am. J. Hum. Genet. 60 (4), 869-878 (1997)    -   PUBMED 9106533-   REFERENCE 20 (bases 1 to 4290)    -   AUTHORS Narahara, K., Baker, E., Ito, S., Yokoyama, Y., Yu, S.,        Hewitt, D., Sutherland, G. R., Eccles, M. R. and Richards, R. I.    -   TITLE Localisation of a 10q breakpoint within the PAX2 gene in a        patient with a de novo t(10;13) translocation and optic nerve        coloboma-renal disease    -   JOURNAL J. Med. Genet. 34 (3), 213-216 (1997)    -   PUBMED 9132492-   REFERENCE 21 (bases 1 to 4290)    -   AUTHORS Dehbi, M., Ghahremani, M., Lechner, M., Dressler, G. and        Pelletier, J.    -   TITLE The paired-box transcription factor, PAX2, positively        modulates expression of the Wilms' tumor suppressor gene (WT1)    -   JOURNAL Oncogene 13 (3), 447-453 (1996)    -   PUBMED 8760285-   REFERENCE 22 (bases 1 to 4290)    -   AUTHORS Sanyanusin, P., Norrish, J. H., Ward, T. A., Nebel, A.,        McNoe, L. A. and Eccles, M. R.    -   TITLE Genomic structure of the human PAX2 gene    -   JOURNAL Genomics 35 (1), 258-261 (1996)    -   PUBMED 8661132-   REFERENCE 23 (bases 1 to 4290)    -   AUTHORS Sanyanusin, P., Schimmenti, L. A., McNoe, L. A.,        Ward, T. A., Pierpont, M. E., Sullivan, M. J., Dobyns, W. B. and        Eccles, M. R.    -   TITLE Mutation of the PAX2 gene in a family with optic nerve        colobomas, renal anomalies and vesicoureteral reflux    -   JOURNAL Nat. Genet. 9 (4), 358-364 (1995)    -   PUBMED 7795640-   REFERENCE 24 (bases 1 to 4290)    -   AUTHORS Ward, T. A., Nebel, A., Reeve, A. E. and Eccles, M. R.    -   TITLE Alternative messenger RNA forms and open reading frames        within an additional conserved region of the human PAX-2 gene    -   JOURNAL Cell Growth Differ. 5 (9), 1015-1021 (1994)    -   PUBMED 7819127-   REFERENCE 25 (bases 1 to 4290)    -   AUTHORS Noll, M.    -   TITLE Evolution and role of Pax genes    -   JOURNAL Curr. Opin. Genet. Dev. 3 (4), 595-605 (1993)    -   PUBMED 8241771    -   REMARK Review article-   REFERENCE 26 (bases 1 to 4290)    -   AUTHORS Stapleton, P., Weith, A., Urbanek, P., Kozmik, Z. and        Busslinger, M.    -   TITLE Chromosomal localization of seven PAX genes and cloning of        a novel family member, PAX-9    -   JOURNAL Nat. Genet. 3 (4), 292-298 (1993)    -   PUBMED 7981748-   REFERENCE 27 (bases 1 to 4290)    -   AUTHORS Pilz, A. J., Povey, S., Gruss, P. and Abbott, C. M.    -   TITLE Mapping of the human homologs of the murine        paired-box-containing genes    -   JOURNAL Mamm. Genome 4 (2), 78-82 (1993)    -   PUBMED 8431641-   REFERENCE 28 (bases 1 to 4290)    -   AUTHORS Eccles, M. R., Wallis, L. J., Fidler, A. E., Spurr, N.        K., Goodfellow, P. J. and Reeve, A. E.    -   TITLE Expression of the PAX2 gene in human fetal kidney and        Wilms' tumor    -   JOURNAL Cell Growth Differ. 3 (5), 279-289 (1992)    -   PUBMED 1378753-   COMMENT REVIEWED REFSEQ: This record has been curated by NCBI staff.    The reference sequence was derived from U45255.1 and BM671839.1. On    Sep. 22, 2003 this sequence version replaced gi:4557824.    -   Summary: PAX2 encodes paired box gene 2, one of many human        homologues of the Drosophila melanogaster gene prd. The central        feature of this transcription factor gene family is the        conserved DNA-binding paired box domain. PAX2 is believed to be        a target of transcriptional supression by the tumor supressor        gene WT1. Mutations within PAX2 have been shown to result in        optic nerve colobomas and renal hypoplasia. Alternative splicing        of this gene results in multiple transcript variants.    -   Transcript Variant: This variant (c) has multiple differences in        the coding region, compared to variant e, one of which results        in a translational frameshift. The resulting protein (isoform c)        has a distinct C-terminus and is shorter than isoform e.    -   COMPLETENESS: complete on the 3′ end.

FEATURES Location/Qualifiers source 1 . . . 4290 /organism = “ Homosapiens” /mol_type = “ mRNA” /db_xref = “ taxon:9606” /chromosome = “10” /map = “ 10q24” gene 1 . . . 4290 /gene = “ PAX2” /db_xref = “GeneID:5076” /db_xref = “ HGNC:8616” /db_xref = “ MIM:167409” CDS 687 .. . 1877 /gene = “ PAX2” /note = “ PAX2 gene is a member of thepaired-box containing genes which encode transcription factors involvedin embryonic and fetal development; the gene product is nuclear proteinwhich binds DNA isoform c is encoded by transcript variant c; paired boxhomeotic gene 2; go_component: nucleus [goid 0005634] [evidence IEA];go_function: ATP binding [goid 0005524] [evidence IEA]; go_function: DNAbinding [goid 0003677] [evidence IEA]; go_function: DNA binding [goid0003677] [evidence TAS] [pmid 9106533]; go_function: nucleosidediphosphate kinase activity [goid 0004550] [evidence IEA]; go_process:development [goid 0007275] [evidence IEA]; go_process: transcription[goid 0006350] [evidence IEA]; go_process: CTP biosynthesis [goid0006241] [evidence IEA]; go_process: GTP biosynthesis [goid 0006183][evidence IEA]; go_process: UTP biosynthesis [goid 0006228] [evidenceIEA]; go_process: axonogenesis [goid 0007409] [evidence TAS] [pmid9106533]; go_process: cell differentiation [goid 0030154] [evidenceIEA]; go_process: visual perception [goid 0007601] [evidence TAS] [pmid9106533]; go_process: regulation of transcription, DNA-dependent [goid0006355] [evidence IEA]; go_process: transcription from RNA polymeraseII promoter [goid 0006366] [evidence TAS] [pmid 8760285]” /codon_start =1 /product = “ paired box protein 2 isoform c” /protein_id = “NP_003979.2” /db_xref = “ GI:34878709” /db_xref = “ CCDS:CCDS7499.1”/db_xref = “ GeneID:5076” /db_xref = “ HGNC:8616” /db_xref = “MIM:167409” /translation = “ MDMHCKADPFSAMHPGHGGVNQLGGVFVNGRPLPDVVRQRIVELAHQGVRPCDISRQLRVSHGCVSKILGRYYETGSIKPGVIGGSKPKVATPKVVDKIAEYKRQNPTMFAWEIRDRLLAEGICDNDTVPSVSSINRIIRTKVQQPFHPTPDGAGTGVTAPGHTIVPSTASPPVSSASNDPVGSYSINGILGIPRSNGEKRKRDEDVSEGSVPNGDSQSGVDSLRKHLRADTFTQQQLEALDRVFERPSYPDVFQASEHIKSEQGNEYSLPALTPGLDEVKSSLSASTNPELGSNVSGTQTYPVVTGRDMASTTLPGYPPHVPPTGQGSYPTSTLAGMVPEAAVGPSSSLMSKPGRKLAEVPPCVQPTGASSPATRTATPSTRPTTRLGDSA (SEQ ID NO: 45) TPPY” STS 1940 . . .2146 /gene = “ PAX2” /standard_name = “ RH80285” /db_xref = “UniSTS:88437” STS 3138 . . . 3290 /gene = “ PAX2” /standard_name = “D10S2478” /db_xref = “ UniSTS:74159” polyA_signal 4244 . . . 4249 /gene= “ PAX2” polyA_signal 4248 . . . 4253 /gene = “ PAX2” polyA_signal 4255. . . 4260 /gene = “ PAX2” polyA_site 4273 /gene = “ PAX2”

-   ORIGIN

(SEQ ID NO: 46)    1 aggctccagt ctccggccga gtcttctcgc agccgcaacccacctggggc cagcccagag   61 ctgccagcgc cgctcggctc cctccctccc tcccggcccttcggccgcgg cggcgtgcgc  121 ctgccttttc cgggggcggg ggcctggccc gcgcgctcccctcccgcagg cgccacctcg  181 gacatccccg ggattgctac ttctctgcca acttcgccaactcgccagca cttggagagg  241 cccggctccc ctcccggcgc cctctgaccg cccccgccccgcgcgctctc cgaccaccgc  301 ctctcggatg accaggttcc aggggagctg agcgagtcgcctcccccgcc cagcttcagc  361 cctggctgca gctgcagcgc gagccatgcg cccccagtgcaccccggccc ggcccaccgc  421 cccggggcca ttctgctgac cgcccagccc cgagccccgacagtggcaag ttgcggctac  481 tgcagttgca agctccggcc aacccggagg agccccagcggggagcgcag tgttgcgccc  541 cccgcccccg cgcgccccgc agcagccggg cgttcactcatcctccctcc cccaccgtcc  601 ctcccttttc tcctcaagtc ctgaagttga gtttgagaggcgacacggcg gcggcggccg  661 cgctgctccc gctcctctgc ctccccatgg atatgcactgcaaagcagac cccttctccg  721 cgatgcaccc agggcacggg ggtgtgaacc agctcgggggggtgtttgtg aacggccggc  781 ccctacccga cgtggtgagg cagcgcatcg tggagctggcccaccagggt gtgcggccct  841 gtgacatctc ccggcagctg cgggtcagcc acggctgtgtcagcaaaatc ctgggcaggt  901 actacgagac cggcagcatc aagccgggtg tgatcggtggctccaagccc aaagtggcga  961 cgcccaaagt ggtggacaag attgctgaat acaaacgacagaacccgact atgttcgcct 1021 gggagattcg agaccggctc ctggccgagg gcatctgtgacaatgacaca gtgcccagcg 1081 tctcttccat caacagaatc atccggacca aagttcagcagcctttccac ccaacgccgg 1141 atggggctgg gacaggagtg accgcccctg gccacaccattgttcccagc acggcctccc 1201 ctcctgtttc cagcgcctcc aatgacccag tgggatcctactccatcaat gggatcctgg 1261 ggattcctcg ctccaatggt gagaagagga aacgtgatgaagatgtgtct gagggctcag 1321 tccccaatgg agattcccag agtggtgtgg acagtttgcggaagcacttg cgagctgaca 1381 ccttcaccca gcagcagctg gaagctttgg atcgggtctttgagcgtcct tcctaccctg 1441 acgtcttcca ggcatcagag cacatcaaat cagaacaggggaacgagtac tccctcccag 1501 ccctgacccc tgggcttgat gaagtcaagt cgagtctatctgcatccacc aaccctgagc 1561 tgggcagcaa cgtgtcaggc acacagacat acccagttgtgactggtcgt gacatggcga 1621 gcaccactct gcctggttac ccccctcacg tgccccccactggccaggga agctacccca 1681 cctccaccct ggcaggaatg gtgcctgagg ctgcagttggtccctcatcc tccctcatga 1741 gcaagccggg gaggaagctt gcagaagtgc ccccttgtgtgcaacccact ggagcgagtt 1801 ctccggcaac ccgtacagcc acccccagta cacggcctacaacgaggctt ggagattcag 1861 caaccccgcc ttactaagtt ccccttatta ttatagtgccgccccccggt ccgcccctgc 1921 cgctgctgcc gctgcctatg accgccacta gttaccgcggggaccacatc aagcttcagg 1981 ccgacagctt cggcctccac atcgtccccg tctgaccccaccccggaggg agggaggacc 2041 gacgcgacgc gatgcctccc ggccaccgcc ccagcctcaccccatcccac gacccccgca 2101 acccttcaca tcacccccct cgaaggtcgg acaggacgggtggagccgtg ggcgggaccc 2161 tcaggcccgg gcccgccgcc cccagccccg cctgccgcccctccccgcct gcctggactg 2221 cgcggcgccg tgagggggat tcggcccagc tcgtcccggcctccaccaag ccagccccga 2281 agcccgccag ccaccctgcc ggactcgggc gcgacctgctggcgcgcgcc ggatgtttct 2341 gtgacacaca atcagcgcgg accgcagcgc ggcccagccccgggcacccg cctcggacgc 2401 tcgggcgcca ggaggcttcg ctggaggggc tgggccaaggagattaagaa gaaaacgact 2461 ttctgcagga ggaagagccc gctgccgaat ccctgggaaaaattcttttc ccccagtgcc 2521 agccggactg ccctcgcctt ccgggtgtgc cctgtcccagaagatggaat gggggtgtgg 2581 gggtccggct ctaggaacgg gctttggggg cgtcaggtctttccaaggtt gggacccaag 2641 gatcgggggg cccagcagcc cgcaccgatc gagccggactctcggctctt cactgctcct 2701 cctggcctgc ctagttcccc agggcccggc acctcctgctgcgagacccg gctctcagcc 2761 ctgccttgcc cctacctcag cgtctcttcc acctgctggcctcccagttt cccctcctgc 2821 cagtccttcg cctgtccctt gacgccctgc atcctcctccctgactcgca gccccatcgg 2881 acgctctccc gggaccgccg caggaccagt ttccatagactgcggactgg ggtcttcctc 2941 cagcagttac ttgatgcccc ctcccccgac acagactctcaatctgccgg tggtaagaac 3001 cggttctgag ctggcgtctg agctgctgcg gggtggaagtggggggctgc ccactccact 3061 cctcccatcc cctcccagcc tcctcctccg gcaggaactgaacagaacca caaaaagtct 3121 acatttattt aatatgatgg tctttgcaaa aaggaacaaaacaacacaaa agcccaccag 3181 gctgctgctt tgtggaaaga cggtgtgtgt cgtgtgaaggcgaaacccgg tgtacataac 3241 ccctccccct ccgccccgcc ccgcccggcc ccgtagagtccctgtcgccc gccggccctg 3301 cctgtagata cgccccgctg tctgtgctgt gagagtcgccgctcgctggg ggggaagggg 3361 gggacacagc tacacgccca ttaaagcaca gcacgtcctgggggaggggg gcatttttta 3421 tgttacaaaa aaaaattacg aaagaaaaga aatctctatgcaaaatgacg aacatggtcc 3481 tgtggactcc tctggcctgt tttgttggct ctttctctgtaattccgtgt tttcgctttt 3541 tcctccctgc ccctctctcc ctctgcccct ctctcctctccgcttctctc cccctctgtc 3601 tctgtctctc tccgtctctg tcgctcttgt ctgtctgtctctgctctttc ctcggcctct 3661 ctccccagac ctggcccggc cgccctgtct ccgcaggctagatccgaggt ggcagctcca 3721 gcccccgggc tcgccccctc gcgggcgtgc cccgcgcgccccgggcggcc gaaggccggg 3781 ccgccccgtc ccgccccgta gttgctcttt cggtagtggcgatgcgccct gcatgtctcc 3841 tcacccgtgg atcgtgacga ctcgaaataa cagaaacaaagtcaataaag tgaaaataaa 3901 taaaaatcct tgaacaaatc cgaaaaggct tggagtcctcgcccagatct ctctcccctg 3961 cgagcccttt ttatttgaga aggaaaaaga gaaaagagaatcgtttaagg gaacccggcg 4021 cccagccagg ctccagtggc ccgaacgggg cggcgagggcggcgagggcg ccgaggtccg 4081 gcccatccca gtcctgtggg gctggccggg cagagaccccggacccaggc ccaggcctaa 4141 cctgctaaat gtccccggac ggttctggtc tcctcggccactttcagtgc gtcggttcgt 4201 tttgattctt tttcttttgt gcacataaga aataaataataataataaat aaagaataaa 4261 attttgtatg tcaaaaaaaa aaaaaaaaaa

Appendix E

-   LOCUS NM_(—)003989 4188 bp mRNA linear PRI Sep. 24, 2005-   DEFINITION Homo sapiens paired box gene 2 (PAX2), transcript variant    d, mRNA.-   ACCESSION NM_(—)003989-   VERSION NM_(—)003989.2 GI:34878715-   KEYWORDS.-   SOURCE Homo sapiens (human)    -   ORGANISM Homo sapiens Eukaryota; Metazoa; Chordata; Craniata;        Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires;        Primates; Catarrhini; Hominidae; Homo.-   REFERENCE 1 (bases 1 to 4188)    -   AUTHORS Yoshimura, K., Yoshida, S., Yamaji, Y., Komori, A.,        Yoshida, A., Hatae, K., Kubota, T. and Ishibashi, T.    -   TITLE De novo insG619 mutation in PAX2 gene in a Japanese        patient with papillorenal syndrome    -   JOURNAL Am. J. Ophthalmol. 139 (4), 733-735 (2005)    -   PUBMED 15808183    -   REMARK GeneRIF: Molecular genetic analysis of the PAX2 gene in        combination with renal ultrasonography can help in making an        earlier diagnosis of the disease.-   REFERENCE 2 (bases 1 to 4188)    -   AUTHORS Mazal, P. R., Stichenwirth, M., Koller, A., Blach, S.,        Haitel, A. and Susani, M.    -   TITLE Expression of aquaporins and PAX-2 compared to CD10 and        cytokeratin 7 in renal neoplasms: a tissue microarray study    -   JOURNAL Mod. Pathol. 18 (4), 535-540 (2005)    -   PUBMED 15502805    -   REMARK GeneRIF: PAX-2 is a reliable marker for clear cell renal        cell carcinomas of lower grades but not for higher grades.-   REFERENCE 3 (bases 1 to 4188)    -   AUTHORS Higashide, T., Wada, T., Sakurai, M., Yokoyama, H. and        Sugiyama, K.    -   TITLE Macular abnormalities and optic disk anomaly associated        with a new PAX2 missense mutation    -   JOURNAL Am. J. Ophthalmol. 139 (1), 203-205 (2005)    -   PUBMED 15652857    -   REMARK GeneRIF: A new PAX2 missense mutation, R71T, may cause        macular abnormalities in addition to anomalies of the optic disk        and the kidney.-   REFERENCE 4 (bases 1 to 4188)    -   AUTHORS Buttiglieri, S., Deregibus, M. C., Bravo, S., Cassoni,        P., Chiarle, R., Bussolati, B. and Camussi, G.    -   TITLE Role of Pax2 in apoptosis resistance and proinvasive        phenotype of Kaposi's sarcoma cells    -   JOURNAL J. Biol. Chem. 279 (6), 4136-4143 (2004)    -   PUBMED 14627715    -   REMARK GeneRIF: expression of Pax2 by Kaposi's sarcoma cells        correlated with an enhanced resistance against apoptotic signals        and with the proinvasive phenotype-   REFERENCE 5 (bases 1 to 4188)    -   AUTHORS Brophy, P. D., Lang, K. M. and Dressler, G. R.    -   TITLE The secreted frizzled related protein 2 (SFRP2) gene is a        target of the Pax2 transcription factor    -   JOURNAL J. Biol. Chem. 278 (52), 52401-52405 (2003)    -   PUBMED 14561758    -   REMARK GeneRIF: Pax2 protein regulates expression of secreted        frizzled related protein 2-   REFERENCE 6 (bases 1 to 4188)    -   AUTHORS Schimmenti, L. A., Manligas, G. S. and Sieving, P. A.    -   TITLE Optic nerve dysplasia and renal insufficiency in a family        with a novel PAX2 mutation, Arg115X: further ophthalmologic        delineation of the renal-coloboma syndrome    -   JOURNAL Ophthalmic Genet. 24 (4), 191-202 (2003)    -   PUBMED 14566649    -   REMARK GeneRIF: PAX2 mutation is associated with Optic nerve        dysplasia and renal insufficiency of the renal-coloboma syndrome-   REFERENCE 7 (bases 1 to 4188)    -   AUTHORS Muratovska, A., Zhou, C., He, S., Goodyer, P. and        Eccles, M. R.    -   TITLE Paired-Box genes are frequently expressed in cancer and        often required for cancer cell survival    -   JOURNAL Oncogene 22 (39), 7989-7997 (2003)    -   PUBMED 12970747    -   REMARK GeneRIF: The PAX2 gene was frequently expressed in a        panel of 406 common primary tumor tissues and endogenous PAX        gene expression is often required for the growth and survival of        cancer cells-   REFERENCE 8 (bases 1 to 4188)    -   AUTHORS Gough, S. M., McDonald, M., Chen, X. N., Korenberg, J.        R., Neri, A., Kahn, T., Eccles, M. R. and Morris, C. M.    -   TITLE Refined physical map of the human PAX2/HOX11/NFKB2 cancer        gene region at 10q24 and relocalization of the HPV6AI1 viral        integration site to 14q13.3-q21.1    -   JOURNAL BMC Genomics 4 (1), 9 (2003)    -   PUBMED 12697057-   REFERENCE 9 (bases 1 to 4188)    -   AUTHORS Hoffmeister, A., Ropolo, A., Vasseur, S., Mallo, G. V.,        Bodeker, H., Ritz-Laser, B., Dressler, G. R., Vaccaro, M. I.,        Dagorn, J. C., Moreno, S. and Iovanna, J. L.    -   TITLE The HMG-I/Y-related protein p8 binds to p300 and Pax2        trans-activation domain-interacting protein to regulate the        trans-activation activity of the Pax2A and Pax2B transcription        factors on the glucagon gene promoter    -   JOURNAL J. Biol. Chem. 277 (25), 22314-22319 (2002)    -   PUBMED 11940591    -   REMARK GeneRIF: The HMG-I/Y-related protein p8 binds to p300 and        Pax2 trans-activation domain-interacting protein to regulate the        trans-activation activity of the Pax2A and Pax2B transcription        factors on the glucagon gene promoter.-   REFERENCE 10 (bases 1 to 4188)    -   AUTHORS Cai, Y., Lechner, M. S., Nihalani, D., Prindle, M. J.,        Holzman, L. B. and Dressler, G. R.    -   TITLE Phosphorylation of Pax2 by the c-Jun N-terminal kinase and        enhanced Pax2-dependent transcription activation    -   JOURNAL J. Biol. Chem. 277 (2), 1217-1222 (2002)    -   PUBMED 11700324-   REFERENCE 11 (bases 1 to 4188)    -   AUTHORS Becker, K., Beales, P. L., Calver, D. M., Matthijs, G.        and Mohammed, S. N.    -   TITLE Okihiro syndrome and acro-renal-ocular syndrome: clinical        overlap, expansion of the phenotype, and absence of PAX2        mutations in two new families    -   JOURNAL J. Med. Genet. 39 (1), 68-71 (2002)    -   PUBMED 11826030    -   REMARK GeneRIF: The absence of PAX2 mutations has been        identified in two families with histories of clinical overlap of        Okihiro and acro-renal-ocular syndromes.-   REFERENCE 12 (bases 1 to 4188)    -   AUTHORS Eccles, M. R., He, S., Legge, M., Kumar, R., Fox, J.,        Zhou, C., French, M. and Tsai, R. W.    -   TITLE PAX genes in development and disease: the role of PAX2 in        urogenital tract development    -   JOURNAL Int. J. Dev. Biol. 46 (4), 535-544 (2002)    -   PUBMED 12141441    -   REMARK Review article GeneRIF: PAX2 has a role in urogenital        tract development and disease [review]-   REFERENCE 13 (bases 1 to 4188)    -   AUTHORS Chung, G. W., Edwards, A. O., Schimmenti, L. A.,        Manligas, G. S., Zhang, Y. H. and Ritter, R. III.    -   TITLE Renal-coloboma syndrome: report of a novel PAX2 gene        mutation    -   JOURNAL Am. J. Ophthalmol. 132 (6), 910-914 (2001)    -   PUBMED 11730657    -   REMARK GeneRIF: The causal relationship between PAX2 gene        mutations and renal-coloboma syndrome is further supported-   REFERENCE 14 (bases 1 to 4188)    -   AUTHORS Nishimoto, K., Iijima, K., Shirakawa, T., Kitagawa, K.,        Satomura, K., Nakamura, H. and Yoshikawa, N.    -   TITLE PAX2 gene mutation in a family with isolated renal        hypoplasia    -   JOURNAL J. Am. Soc. Nephrol. 12 (8), 1769-1772 (2001)    -   PUBMED 11461952-   REFERENCE 15 (bases 1 to 4188)    -   AUTHORS Ritz-Laser, B., Estreicher, A., Gauthier, B. and        Philippe, J.    -   TITLE The paired homeodomain transcription factor Pax-2 is        expressed in the endocrine pancreas and transactivates the        glucagon gene promoter    -   JOURNAL J. Biol. Chem. 275 (42), 32708-32715 (2000)    -   PUBMED 10938089-   REFERENCE 16 (bases 1 to 4188)    -   AUTHORS Lechner, M. S., Levitan, I. and Dressler, G. R.    -   TITLE PTIP, a novel BRCT domain-containing protein interacts        with Pax2 and is associated with active chromatin    -   JOURNAL Nucleic Acids Res. 28 (14), 2741-2751 (2000)    -   PUBMED 10908331-   REFERENCE 17 (bases 1 to 4188)    -   AUTHORS Tavassoli, K., Ruger, W. and Horst, J.    -   TITLE Alternative splicing in PAX2 generates a new reading frame        and an extended conserved coding region at the carboxy terminus    -   JOURNAL Hum. Genet. 101 (3), 371-375 (1997)    -   PUBMED 9439670-   REFERENCE 18 (bases 1 to 4188)    -   AUTHORS Dahl, E., Koseki, H. and Balling, R.    -   TITLE Pax genes and organogenesis    -   JOURNAL Bioessays 19 (9), 755-765 (1997)    -   PUBMED 9297966    -   REMARK Review article-   REFERENCE 19 (bases 1 to 4188)    -   AUTHORS Schimmenti, L. A., Cunliffe, H. E., McNoe, L. A.,        Ward, T. A., French, M. C., Shim, H. H., Zhang, Y. H.,        Proesmans, W., Leys, A., Byerly, K. A., Braddock, S. R., Masuno,        M., Imaizumi, K., Devriendt, K. and Eccles, M. R.    -   TITLE Further delineation of renal-coloboma syndrome in patients        with extreme variability of phenotype and identical PAX2        mutations    -   JOURNAL Am. J. Hum. Genet. 60 (4), 869-878 (1997)    -   PUBMED 9106533-   REFERENCE 20 (bases 1 to 4188)    -   AUTHORS Narahara, K., Baker, E., Ito, S., Yokoyama, Y., Yu, S.,        Hewitt, D., Sutherland, G. R., Eccles, M. R. and Richards, R. I.    -   TITLE Localisation of a 10q breakpoint within the PAX2 gene in a        patient with a de novo t(10;13) translocation and optic nerve        coloboma-renal disease    -   JOURNAL J. Med. Genet. 34 (3), 213-216 (1997)    -   PUBMED 9132492-   REFERENCE 21 (bases 1 to 4188)    -   AUTHORS Dehbi, M., Ghahremani, M., Lechner, M., Dressler, G. and        Pelletier, J.    -   TITLE The paired-box transcription factor, PAX2, positively        modulates expression of the Wilms' tumor suppressor gene (WT1)    -   JOURNAL Oncogene 13 (3), 447-453 (1996)    -   PUBMED 8760285-   REFERENCE 22 (bases 1 to 4188)    -   AUTHORS Sanyanusin, P., Norrish, J. H., Ward, T. A., Nebel, A.,        McNoe, L. A. and Eccles, M. R.    -   TITLE Genomic structure of the human PAX2 gene    -   JOURNAL Genomics 35 (1), 258-261 (1996)    -   PUBMED 8661132-   REFERENCE 23 (bases 1 to 4188)    -   AUTHORS Sanyanusin, P., Schimmenti, L. A., McNoe, L. A.,        Ward, T. A., Pierpont, M. E., Sullivan, M. J., Dobyns, W. B. and        Eccles, M. R.    -   TITLE Mutation of the PAX2 gene in a family with optic nerve        colobomas, renal anomalies and vesicoureteral reflux    -   JOURNAL Nat. Genet. 9 (4), 358-364 (1995)    -   PUBMED 7795640-   REFERENCE 24 (bases 1 to 4188)    -   AUTHORS Ward, T. A., Nebel, A., Reeve, A. E. and Eccles, M. R.    -   TITLE Alternative messenger RNA forms and open reading frames        within an additional conserved region of the human PAX-2 gene    -   JOURNAL Cell Growth Differ. 5 (9), 1015-1021 (1994)    -   PUBMED 7819127-   REFERENCE 25 (bases 1 to 4188)    -   AUTHORS Noll, M.    -   TITLE Evolution and role of Pax genes    -   JOURNAL Curr. Opin. Genet. Dev. 3 (4), 595-605 (1993)    -   PUBMED 8241771    -   REMARK Review article-   REFERENCE 26 (bases 1 to 4188)    -   AUTHORS Stapleton, P., Weith, A., Urbanek, P., Kozmik, Z. and        Busslinger, M.    -   TITLE Chromosomal localization of seven PAX genes and cloning of        a novel family member, PAX-9    -   JOURNAL Nat. Genet. 3 (4), 292-298 (1993)    -   PUBMED 7981748-   REFERENCE 27 (bases 1 to 4188)    -   AUTHORS Pilz, A. J., Povey, S., Gruss, P. and Abbott, C. M.    -   TITLE Mapping of the human homologs of the murine        paired-box-containing genes    -   JOURNAL Mamm. Genome 4 (2), 78-82 (1993)    -   PUBMED 8431641-   REFERENCE 28 (bases 1 to 4188)    -   AUTHORS Eccles, M. R., Wallis, L. J., Fidler, A. E., Spurr, N.        K., Goodfellow, P. J. and Reeve, A. E.    -   TITLE Expression of the PAX2 gene in human fetal kidney and        Wilms' tumor    -   JOURNAL Cell Growth Differ. 3 (5), 279-289 (1992)    -   PUBMED 1378753-   COMMENT REVIEWED REFSEQ: This record has been curated by NCBI staff.    The reference sequence was derived from U45255.1 and BM671839.1. On    Sep. 22, 2003 this sequence version replaced gi:4557826.    -   Summary: PAX2 encodes paired box gene 2, one of many human        homologues of the Drosophila melanogaster gene prd. The central        feature of this transcription factor gene family is the        conserved DNA-binding paired box domain. PAX2 is believed to be        a target of transcriptional supression by the tumor supressor        gene WT1. Mutations within PAX2 have been shown to result in        optic nerve colobomas and renal hypoplasia. Alternative splicing        of this gene results in multiple transcript variants.    -   Transcript Variant: This variant (d) lacks an alternate in-frame        exon compared to variant e. This results in an isoform        (isoform d) that is shorter than isoform e.    -   COMPLETENESS: complete on the 3′ end.

FEATURES Location/Qualifiers source 1 . . . 4188 /organism = “ Homosapiens” /mol_type = “ mRNA” /db_xref = “ taxon:9606” /chromosome = “10” /map = “ 10q24” gene 1 . . . 4188 /gene = “ PAX2” /db_xref = “GeneID:5076” /db_xref = “ HGNC:8616” /db_xref = “ HPRD:HPRD_01330”/db_xref = “ MIM:167409” CDS 687 . . . 1913 /gene = “ PAX2” /note = “PAX2 gene is a member of the paired-box containing genes which encodetranscription factors involved in embryonic and fetal development; thegene product is nuclear protein which binds DNA isoform d is encoded bytranscript variant d; paired box homeotic gene 2; go_component: nucleus[goid 0005634] [evidence IEA]; go_function: ATP binding [goid 0005524][evidence IEA]; go_function: DNA binding [goid 0003677] [evidence IEA];go_function: DNA binding [goid 0003677] [evidence TAS] [pmid 9106533];go_function: nucleoside diphosphate kinase activity [goid 0004550][evidence IEA]; go_process: development [goid 0007275] [evidence IEA];go_process: transcription [goid 0006350] [evidence IEA]; go_process: CTPbiosynthesis [goid 0006241] [evidence IEA]; go_process: GTP biosynthesis[goid 0006183] [evidence IEA]; go_process: UTP biosynthesis [goid0006228] [evidence IEA]; go_process: axonogenesis [goid 0007409][evidence TAS] [pmid 9106533]; go_process: cell differentiation [goid0030154] [evidence IEA]; go_process: visual perception [goid 0007601][evidence TAS] [pmid 9106533]; go_process: regulation of transcription,DNA-dependent [goid 0006355] [evidence IEA]; go_process: transcriptionfrom RNA polymerase II promoter [goid 0006366] [evidence TAS] [pmid8760285]” /codon_start = 1 /product = “ paired box protein 2 isoform d”/protein_id = “ NP_003980.2” /db_xref = “ GI:34878716” /db_xref = “GeneID:5076” /db_xref = “ HGNC:8616” /db_xref = “ HPRD:HPRD_01330”/db_xref = “ MIM:167409” /translation = “MDMHCKADPFSAMHPGHGGVNQLGGVFVNGRPLPDVVRQRIVELAHQGVRPCDISRQLRVSHGCVSKILGRYYETGSIKPGVIGGSKPKVATPKVVDKIAEYKRQNPTMFAWEIRDRLLAEGICDNDTVPSVSSINRIIRTKVQQPFHPTPDGAGTGVTAPGHTIVPSTASPPVSSASNDPVGSYSINGILGIPRSNGEKRKRDEDVSEGSVPNGDSQSGVDSLRKHLRADTFTQQQLEALDRVFERPSYPDVFQASEHIKSEQGNEYSLPALTPGLDEVKSSLSASTNPELGSNVSGTQTYPVVTGRDMASTTLPGYPPHVPPTGQGSYPTSTLAGMVPGSEFSGNPYSHPQYTAYNEAWRFSNPALLMPPPGPPLPLLP LPMTATSYRGDHIKLQADSFGLHIVPV”(SEQ ID NO: 47) STS 1838 . . . 2044 /gene = “ PAX2” /standard_name = “RH80285” /db_xref = “ UniSTS:88437” STS 3036 . . . 3188 /gene = “ PAX2”/standard_name = “ D10S2478” /db_xref = “ UniSTS:74159” polyA_signal4142 . . . 4147 /gene = “ PAX2” polyA_signal 4146 . . . 4151 /gene = “PAX2” polyA_signal 4153 . . . 4158 /gene = “ PAX2” polyA_site 4171 /gene= “ PAX2”

-   ORIGIN

(SEQ ID NO: 48)    1 aggctccagt ctccggccga gtcttctcgc agccgcaacccacctggggc cagcccagag   61 ctgccagcgc cgctcggctc cctccctccc tcccggcccttcggccgcgg cggcgtgcgc  121 ctgccttttc cgggggcggg ggcctggccc gcgcgctcccctcccgcagg cgccacctcg  181 gacatccccg ggattgctac ttctctgcca acttcgccaactcgccagca cttggagagg  241 cccggctccc ctcccggcgc cctctgaccg cccccgccccgcgcgctctc cgaccaccgc  301 ctctcggatg accaggttcc aggggagctg agcgagtcgcctcccccgcc cagcttcagc  361 cctggctgca gctgcagcgc gagccatgcg cccccagtgcaccccggccc ggcccaccgc  421 cccggggcca ttctgctgac cgcccagccc cgagccccgacagtggcaag ttgcggctac  481 tgcagttgca agctccggcc aacccggagg agccccagcggggagcgcag tgttgcgccc  541 cccgcccccg cgcgccccgc agcagccggg cgttcactcatcctccctcc cccaccgtcc  601 ctcccttttc tcctcaagtc ctgaagttga gtttgagaggcgacacggcg gcggcggccg  661 cgctgctccc gctcctctgc ctccccatgg atatgcactgcaaagcagac cccttctccg  721 cgatgcaccc agggcacggg ggtgtgaacc agctcgggggggtgtttgtg aacggccggc  781 ccctacccga cgtggtgagg cagcgcatcg tggagctggcccaccagggt gtgcggccct  841 gtgacatctc ccggcagctg cgggtcagcc acggctgtgtcagcaaaatc ctgggcaggt  901 actacgagac cggcagcatc aagccgggtg tgatcggtggctccaagccc aaagtggcga  961 cgcccaaagt ggtggacaag attgctgaat acaaacgacagaacccgact atgttcgcct 1021 gggagattcg agaccggctc ctggccgagg gcatctgtgacaatgacaca gtgcccagcg 1081 tctcttccat caacagaatc atccggacca aagttcagcagcctttccac ccaacgccgg 1141 atggggctgg gacaggagtg accgcccctg gccacaccattgttcccagc acggcctccc 1201 ctcctgtttc cagcgcctcc aatgacccag tgggatcctactccatcaat gggatcctgg 1261 ggattcctcg ctccaatggt gagaagagga aacgtgatgaagatgtgtct gagggctcag 1321 tccccaatgg agattcccag agtggtgtgg acagtttgcggaagcacttg cgagctgaca 1381 ccttcaccca gcagcagctg gaagctttgg atcgggtctttgagcgtcct tcctaccctg 1441 acgtcttcca ggcatcagag cacatcaaat cagaacaggggaacgagtac tccctcccag 1501 ccctgacccc tgggcttgat gaagtcaagt cgagtctatctgcatccacc aaccctgagc 1561 tgggcagcaa cgtgtcaggc acacagacat acccagttgtgactggtcgt gacatggcga 1621 gcaccactct gcctggttac ccccctcacg tgccccccactggccaggga agctacccca 1681 cctccaccct ggcaggaatg gtgcctggga gcgagttctccggcaacccg tacagccacc 1741 cccagtacac ggcctacaac gaggcttgga gattcagcaaccccgcctta ctaatgccgc 1801 cccccggtcc gcccctgccg ctgctgccgc tgcctatgaccgccactagt taccgcgggg 1861 accacatcaa gcttcaggcc gacagcttcg gcctccacatcgtccccgtc tgaccccacc 1921 ccggagggag ggaggaccga cgcgacgcga tgcctcccggccaccgcccc agcctcaccc 1981 catcccacga cccccgcaac ccttcacatc acccccctcgaaggtcggac aggacgggtg 2041 gagccgtggg cgggaccctc aggcccgggc ccgccgcccccagccccgcc tgccgcccct 2101 ccccgcctgc ctggactgcg cggcgccgtg agggggattcggcccagctc gtcccggcct 2161 ccaccaagcc agccccgaag cccgccagcc accctgccggactcgggcgc gacctgctgg 2221 cgcgcgccgg atgtttctgt gacacacaat cagcgcggaccgcagcgcgg cccagccccg 2281 ggcacccgcc tcggacgctc gggcgccagg aggcttcgctggaggggctg ggccaaggag 2341 attaagaaga aaacgacttt ctgcaggagg aagagcccgctgccgaatcc ctgggaaaaa 2401 ttcttttccc ccagtgccag ccggactgcc ctcgccttccgggtgtgccc tgtcccagaa 2461 gatggaatgg gggtgtgggg gtccggctct aggaacgggctttgggggcg tcaggtcttt 2521 ccaaggttgg gacccaagga tcggggggcc cagcagcccgcaccgatcga gccggactct 2581 cggctcttca ctgctcctcc tggcctgcct agttccccagggcccggcac ctcctgctgc 2641 gagacccggc tctcagccct gccttgcccc tacctcagcgtctcttccac ctgctggcct 2701 cccagtttcc cctcctgcca gtccttcgcc tgtcccttgacgccctgcat cctcctccct 2761 gactcgcagc cccatcggac gctctcccgg gaccgccgcaggaccagttt ccatagactg 2821 cggactgggg tcttcctcca gcagttactt gatgccccctcccccgacac agactctcaa 2881 tctgccggtg gtaagaaccg gttctgagct ggcgtctgagctgctgcggg gtggaagtgg 2941 ggggctgccc actccactcc tcccatcccc tcccagcctcctcctccggc aggaactgaa 3001 cagaaccaca aaaagtctac atttatttaa tatgatggtctttgcaaaaa ggaacaaaac 3061 aacacaaaag cccaccaggc tgctgctttg tggaaagacggtgtgtgtcg tgtgaaggcg 3121 aaacccggtg tacataaccc ctccccctcc gccccgccccgcccggcccc gtagagtccc 3181 tgtcgcccgc cggccctgcc tgtagatacg ccccgctgtctgtgctgtga gagtcgccgc 3241 tcgctggggg ggaagggggg gacacagcta cacgcccattaaagcacagc acgtcctggg 3301 ggaggggggc attttttatg ttacaaaaaa aaattacgaaagaaaagaaa tctctatgca 3361 aaatgacgaa catggtcctg tggactcctc tggcctgttttgttggctct ttctctgtaa 3421 ttccgtgttt tcgctttttc ctccctgccc ctctctccctctgcccctct ctcctctccg 3481 cttctctccc cctctgtctc tgtctctctc cgtctctgtcgctcttgtct gtctgtctct 3541 gctctttcct cggcctctct ccccagacct ggcccggccgccctgtctcc gcaggctaga 3601 tccgaggtgg cagctccagc ccccgggctc gccccctcgcgggcgtgccc cgcgcgcccc 3661 gggcggccga aggccgggcc gccccgtccc gccccgtagttgctctttcg gtagtggcga 3721 tgcgccctgc atgtctcctc acccgtggat cgtgacgactcgaaataaca gaaacaaagt 3781 caataaagtg aaaataaata aaaatccttg aacaaatccgaaaaggcttg gagtcctcgc 3841 ccagatctct ctcccctgcg agcccttttt atttgagaaggaaaaagaga aaagagaatc 3901 gtttaaggga acccggcgcc cagccaggct ccagtggcccgaacggggcg gcgagggcgg 3961 cgagggcgcc gaggtccggc ccatcccagt cctgtggggctggccgggca gagaccccgg 4021 acccaggccc aggcctaacc tgctaaatgt ccccggacggttctggtctc ctcggccact 4081 ttcagtgcgt cggttcgttt tgattctttt tcttttgtgcacataagaaa taaataataa 4141 taataaataa agaataaaat tttgtatgtc aaaaaaaaaaaaaaaaaa

Appendix F

-   LOCUS NM_(—)003990 4257 bp mRNA linear PRI Sep. 24, 2005-   DEFINITION Homo sapiens paired box gene 2 (PAX2), transcript variant    e, mRNA.-   ACCESSION NM_(—)003990-   VERSION NM_(—)003990.2 GI:34878702-   KEYWORDS.-   SOURCE Homo sapiens (human)    -   ORGANISM Homo sapiens Eukaryota; Metazoa; Chordata; Craniata;        Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires;        Primates; Catarrhini; Hominidae; Homo.-   REFERENCE 1 (bases 1 to 4257)    -   AUTHORS Yoshimura, K., Yoshida, S., Yamaji, Y., Komori, A.,        Yoshida, A., Hatae, K., Kubota, T. and Ishibashi, T.    -   TITLE De novo insG619 mutation in PAX2 gene in a Japanese        patient with papillorenal syndrome    -   JOURNAL Am. J. Ophthalmol. 139 (4), 733-735 (2005)    -   PUBMED 15808183    -   REMARK GeneRIF: Molecular genetic analysis of the PAX2 gene in        combination with renal ultrasonography can help in making an        earlier diagnosis of the disease.-   REFERENCE 2 (bases 1 to 4257)    -   AUTHORS Mazal, P. R., Stichenwirth, M., Koller, A., Blach, S.,        Haitel, A. and Susani, M.    -   TITLE Expression of aquaporins and PAX-2 compared to CD10 and        cytokeratin 7 in renal neoplasms: a tissue microarray study    -   JOURNAL Mod. Pathol. 18 (4), 535-540 (2005)    -   PUBMED 15502805    -   REMARK GeneRIF: PAX-2 is a reliable marker for clear cell renal        cell carcinomas of lower grades but not for higher grades.-   REFERENCE 3 (bases 1 to 4257)    -   AUTHORS Higashide, T., Wada, T., Sakurai, M., Yokoyama, H. and        Sugiyama, K.    -   TITLE Macular abnormalities and optic disk anomaly associated        with a new PAX2 missense mutation    -   JOURNAL Am. J. Ophthalmol. 139 (1), 203-205 (2005)    -   PUBMED 15652857    -   REMARK GeneRIF: A new PAX2 missense mutation, R71T, may cause        macular abnormalities in addition to anomalies of the optic disk        and the kidney.-   REFERENCE 4 (bases 1 to 4257)    -   AUTHORS Buttiglieri, S., Deregibus, M. C., Bravo, S., Cassoni,        P., Chiarle, R., Bussolati, B. and Camussi, G.    -   TITLE Role of Pax2 in apoptosis resistance and proinvasive        phenotype of Kaposi's sarcoma cells    -   JOURNAL J. Biol. Chem. 279 (6), 4136-4143 (2004)    -   PUBMED 14627715    -   REMARK GeneRIF: expression of Pax2 by Kaposi's sarcoma cells        correlated with an enhanced resistance against apoptotic signals        and with the proinvasive phenotype-   REFERENCE 5 (bases 1 to 4257)    -   AUTHORS Brophy, P. D., Lang, K. M. and Dressler, G. R.    -   TITLE The secreted frizzled related protein 2 (SFRP2) gene is a        target of the Pax2 transcription factor    -   JOURNAL J. Biol. Chem. 278 (52), 52401-52405 (2003)    -   PUBMED 14561758    -   REMARK GeneRIF: Pax2 protein regulates expression of secreted        frizzled related protein 2-   REFERENCE 6 (bases 1 to 4257)    -   AUTHORS Schimmenti, L. A., Manligas, G. S. and Sieving, P. A.    -   TITLE Optic nerve dysplasia and renal insufficiency in a family        with a novel PAX2 mutation, Arg115X: further ophthalmologic        delineation of the renal-coloboma syndrome    -   JOURNAL Ophthalmic Genet. 24 (4), 191-202 (2003)    -   PUBMED 14566649    -   REMARK GeneRIF: PAX2 mutation is associated with Optic nerve        dysplasia and renal insufficiency of the renal-coloboma syndrome-   REFERENCE 7 (bases 1 to 4257)    -   AUTHORS Muratovska, A., Zhou, C., He, S., Goodyer, P. and        Eccles, M. R.    -   TITLE Paired-Box genes are frequently expressed in cancer and        often required for cancer cell survival    -   JOURNAL Oncogene 22 (39), 7989-7997 (2003)    -   PUBMED 12970747    -   REMARK GeneRIF: The PAX2 gene was frequently expressed in a        panel of 406 common primary tumor tissues and endogenous PAX        gene expression is often required for the growth and survival of        cancer cells-   REFERENCE 8 (bases 1 to 4257)    -   AUTHORS Gough, S. M., McDonald, M., Chen, X. N., Korenberg, J.        R., Neri, A., Kahn, T., Eccles, M. R. and Morris, C. M.    -   TITLE Refined physical map of the human PAX2/HOX11/NFKB2 cancer        gene region at 10q24 and relocalization of the HPV6AI1 viral        integration site to 14q13.3-q21.1    -   JOURNAL BMC Genomics 4 (1), 9 (2003)    -   PUBMED 12697057-   REFERENCE 9 (bases 1 to 4257)    -   AUTHORS Hoffmeister, A., Ropolo, A., Vasseur, S., Mallo, G. V.,        Bodeker, H., Ritz-Laser, B., Dressler, G. R., Vaccaro, M. I.,        Dagorn, J. C., Moreno, S. and Iovanna, J. L.    -   TITLE The HMG-IN-related protein p8 binds to p300 and Pax2        trans-activation domain-interacting protein to regulate the        trans-activation activity of the Pax2A and Pax2B transcription        factors on the glucagon gene promoter    -   JOURNAL J. Biol. Chem. 277 (25), 22314-22319 (2002)    -   PUBMED 11940591    -   REMARK GeneRIF: The HMG-I/Y-related protein p8 binds to p300 and        Pax2 trans-activation domain-interacting protein to regulate the        trans-activation activity of the Pax2A and Pax2B transcription        factors on the glucagon gene promoter.-   REFERENCE 10 (bases 1 to 4257)    -   AUTHORS Cai, Y., Lechner, M. S., Nihalani, D., Prindle, M. J.,        Holzman, L. B. and Dressler, G. R.    -   TITLE Phosphorylation of Pax2 by the c-Jun N-terminal kinase and        enhanced Pax2-dependent transcription activation    -   JOURNAL J. Biol. Chem. 277 (2), 1217-1222 (2002)    -   PUBMED 11700324-   REFERENCE 11 (bases 1 to 4257)    -   AUTHORS Becker, K., Beales, P. L., Calver, D. M., Matthijs, G.        and Mohammed, S. N.    -   TITLE Okihiro syndrome and acro-renal-ocular syndrome: clinical        overlap, expansion of the phenotype, and absence of PAX2        mutations in two new families    -   JOURNAL J. Med. Genet. 39 (1), 68-71 (2002)    -   PUBMED 11826030    -   REMARK GeneRIF: The absence of PAX2 mutations has been        identified in two families with histories of clinical overlap of        Okihiro and acro-renal-ocular syndromes.-   REFERENCE 12 (bases 1 to 4257)    -   AUTHORS Eccles, M. R., He, S., Legge, M., Kumar, R., Fox, J.,        Zhou, C., French, M. and Tsai, R. W.    -   TITLE PAX genes in development and disease: the role of PAX2 in        urogenital tract development    -   JOURNAL Int. J. Dev. Biol. 46 (4), 535-544 (2002)    -   PUBMED 12141441    -   REMARK Review article GeneRIF: PAX2 has a role in urogenital        tract development and disease [review]-   REFERENCE 13 (bases 1 to 4257)    -   AUTHORS Chung, G. W., Edwards, A. O., Schimmenti, L. A.,        Manligas, G. S., Zhang, Y. H. and Ritter, R. III.    -   TITLE Renal-coloboma syndrome: report of a novel PAX2 gene        mutation    -   JOURNAL Am. J. Ophthalmol. 132 (6), 910-914 (2001)    -   PUBMED 11730657    -   REMARK GeneRIF: The causal relationship between PAX2 gene        mutations and renal-coloboma syndrome is further supported-   REFERENCE 14 (bases 1 to 4257)    -   AUTHORS Nishimoto, K., Iijima, K., Shirakawa, T., Kitagawa, K.,        Satomura, K., Nakamura, H. and Yoshikawa, N.    -   TITLE PAX2 gene mutation in a family with isolated renal        hypoplasia    -   JOURNAL J. Am. Soc. Nephrol. 12 (8), 1769-1772 (2001)    -   PUBMED 11461952-   REFERENCE 15 (bases 1 to 4257)    -   AUTHORS Ritz-Laser, B., Estreicher, A., Gauthier, B. and        Philippe, J.    -   TITLE The paired homeodomain transcription factor Pax-2 is        expressed in the endocrine pancreas and transactivates the        glucagon gene promoter    -   JOURNAL J. Biol. Chem. 275 (42), 32708-32715 (2000)    -   PUBMED 10938089-   REFERENCE 16 (bases 1 to 4257)    -   AUTHORS Lechner, M. S., Levitan, I. and Dressler, G. R.    -   TITLE PTIP, a novel BRCT domain-containing protein interacts        with Pax2 and is associated with active chromatin    -   JOURNAL Nucleic Acids Res. 28 (14), 2741-2751 (2000)    -   PUBMED 10908331-   REFERENCE 17 (bases 1 to 4257)    -   AUTHORS Tavassoli, K., Ruger, W. and Horst, J.    -   TITLE Alternative splicing in PAX2 generates a new reading frame        and an extended conserved coding region at the carboxy terminus    -   JOURNAL Hum. Genet. 101 (3), 371-375 (1997)    -   PUBMED 9439670-   REFERENCE 18 (bases 1 to 4257)    -   AUTHORS Dahl, E., Koseki, H. and Balling, R.    -   TITLE Pax genes and organogenesis    -   JOURNAL Bioessays 19 (9), 755-765 (1997)    -   PUBMED 9297966    -   REMARK Review article-   REFERENCE 19 (bases 1 to 4257)    -   AUTHORS Schimmenti, L. A., Cunliffe, H. E., McNoe, L. A.,        Ward, T. A., French, M. C., Shim, H. H., Zhang, Y. H.,        Proesmans, W., Leys, A., Byerly, K. A., Braddock, S. R., Masuno,        M., Imaizumi, K., Devriendt, K. and Eccles, M. R.    -   TITLE Further delineation of renal-coloboma syndrome in patients        with extreme variability of phenotype and identical PAX2        mutations    -   JOURNAL Am. J. Hum. Genet. 60 (4), 869-878 (1997)    -   PUBMED 9106533-   REFERENCE 20 (bases 1 to 4257)    -   AUTHORS Narahara, K., Baker, E., Ito, S., Yokoyama, Y., Yu, S.,        Hewitt, D., Sutherland, G. R., Eccles, M. R. and Richards, R. I.    -   TITLE Localisation of a 10q breakpoint within the PAX2 gene in a        patient with a de novo t(10;13) translocation and optic nerve        coloboma-renal disease    -   JOURNAL J. Med. Genet. 34 (3), 213-216 (1997)    -   PUBMED 9132492-   REFERENCE 21 (bases 1 to 4257)    -   AUTHORS Dehbi, M., Ghahremani, M., Lechner, M., Dressler, G. and        Pelletier, J.    -   TITLE The paired-box transcription factor, PAX2, positively        modulates expression of the Wilms' tumor suppressor gene (WT1)    -   JOURNAL Oncogene 13 (3), 447-453 (1996)    -   PUBMED 8760285-   REFERENCE 22 (bases 1 to 4257)    -   AUTHORS Sanyanusin, P., Norrish, J. H., Ward, T. A., Nebel, A.,        McNoe, L. A. and Eccles, M. R.    -   TITLE Genomic structure of the human PAX2 gene    -   JOURNAL Genomics 35 (1), 258-261 (1996)    -   PUBMED 8661132-   REFERENCE 23 (bases 1 to 4257)    -   AUTHORS Sanyanusin, P., Schimmenti, L. A., McNoe, L. A.,        Ward, T. A., Pierpont, M. E., Sullivan, M. J., Dobyns, W. B. and        Eccles, M. R.    -   TITLE Mutation of the PAX2 gene in a family with optic nerve        colobomas, renal anomalies and vesicoureteral reflux    -   JOURNAL Nat. Genet. 9 (4), 358-364 (1995)    -   PUBMED 7795640-   REFERENCE 24 (bases 1 to 4257)    -   AUTHORS Ward, T. A., Nebel, A., Reeve, A. E. and Eccles, M. R.    -   TITLE Alternative messenger RNA forms and open reading frames        within an additional conserved region of the human PAX-2 gene    -   JOURNAL Cell Growth Differ. 5 (9), 1015-1021 (1994)    -   PUBMED 7819127-   REFERENCE 25 (bases 1 to 4257)    -   AUTHORS Noll, M.    -   TITLE Evolution and role of Pax genes    -   JOURNAL Curr. Opin. Genet. Dev. 3 (4), 595-605 (1993)    -   PUBMED 8241771    -   REMARK Review article-   REFERENCE 26 (bases 1 to 4257)    -   AUTHORS Stapleton, P., Weith, A., Urbanek, P., Kozmik, Z. and        Busslinger, M.    -   TITLE Chromosomal localization of seven PAX genes and cloning of        a novel family member, PAX-9    -   JOURNAL Nat. Genet. 3 (4), 292-298 (1993)    -   PUBMED 7981748-   REFERENCE 27 (bases 1 to 4257)    -   AUTHORS Pilz, A. J., Povey, S., Gruss, P. and Abbott, C. M.    -   TITLE Mapping of the human homologs of the murine        paired-box-containing genes    -   JOURNAL Mamm. Genome 4 (2), 78-82 (1993)    -   PUBMED 8431641-   REFERENCE 28 (bases 1 to 4257)    -   AUTHORS Eccles, M. R., Wallis, L. J., Fidler, A. E., Spurr, N.        K., Goodfellow, P. J. and Reeve, A. E.    -   TITLE Expression of the PAX2 gene in human fetal kidney and        Wilms' tumor    -   JOURNAL Cell Growth Differ. 3 (5), 279-289 (1992)    -   PUBMED 1378753-   COMMENT REVIEWED REFSEQ: This record has been curated by NCBI staff.    The reference sequence was derived from U45255.1 and BM671839.1. On    Sep. 22, 2003 this sequence version replaced gi:4557828.    -   Summary: PAX2 encodes paired box gene 2, one of many human        homologues of the Drosophila melanogaster gene prd. The central        feature of this transcription factor gene family is the        conserved DNA-binding paired box domain. PAX2 is believed to be        a target of transcriptional supression by the tumor supressor        gene WT1. Mutations within PAX2 have been shown to result in        optic nerve colobomas and renal hypoplasia. Alternative splicing        of this gene results in multiple transcript variants.    -   Transcript Variant: This variant (e) encodes the longest isoform        (e).    -   COMPLETENESS: complete on the 3′ end.

FEATURES Location/Qualifiers source 1 . . . 4257 /organism = “ Homosapiens” /mol_type = “ mRNA” /db_xref = “ taxon:9606” /chromosome = “10” /map = “ 10q24” gene 1 . . . 4257 /gene = “ PAX2” /db_xref = “GeneID:5076” /db_xref = “ HGNC:8616” /db_xref = “ MIM:167409” CDS 687 .. . 1982 /gene = “ PAX2” /note = “ PAX2 gene is a member of thepaired-box containing genes which encode transcription factors involvedin embryonic and fetal development; the gene product is nuclear proteinwhich binds DNA isoform e is encoded by transcript variant e; paired boxhomeotic gene 2; go_component: nucleus [goid 0005634] [evidence IEA];go_function: ATP binding [goid 0005524] [evidence IEA]; go_function: DNAbinding [goid 0003677] [evidence IEA]; go_function: DNA binding [goid0003677] [evidence TAS] [pmid 9106533]; go_function: nucleosidediphosphate kinase activity [goid 0004550] [evidence IEA]; go_process:development [goid 0007275] [evidence IEA]; go_process: transcription[goid 0006350] [evidence IEA]; go_process: CTP biosynthesis [goid0006241] [evidence IEA]; go_process: GTP biosynthesis [goid 0006183][evidence IEA]; go_process: UTP biosynthesis [goid 0006228] [evidenceIEA]; go_process: axonogenesis [goid 0007409] [evidence TAS] [pmid9106533]; go_process: cell differentiation [goid 0030154] [evidenceIEA]; go_process: visual perception [goid 0007601] [evidence TAS] [pmid9106533]; go_process: regulation of transcription, DNA-dependent [goid0006355] [evidence IEA]; go_process: transcription from RNA polymeraseII promoter [goid 0006366] [evidence TAS] [pmid 8760285]” /codon_start =1 /product = “ paired box protein 2 isoform e” /protein_id = “ NP003981.2” /db_xref = “ GI:34878703” /db_xref = “ GeneID:5076” /db_xref =“ HGNC:8616” /db_xref = “ MIM:167409” /translation = “MDMHCKADPFSAMHPGHGGVNQLGGVFVNGRPLPDVVRQRIVELAHQGVRPCDISRQLRVSHGCVSKILGRYYETGSIKPGVIGGSKPKVATPKVVDKIAEYKRQNPTMFAWEIRDRLLAEGICDNDTVPSVSSINRIIRTKVQQPFHPTPDGAGTGVTAPGHTIVPSTASPPVSSASNDPVGSYSINGILGIPRSNGEKRKRDEVEVYTDPAHIRGGGGLHLVWTLRDVSEGSVPNGDSQSGVDSLRKHLRADTFTQQQLEALDRVFERPSYPDVFQASEHIKSEQGNEYSLPALTPGLDEVKSSLSASTNPELGSNVSGTQTYPVVTGRDMASTTLPGYPPHVPPTGQGSYPTSTLAGMVPGSEFSGNPYSHPQYTAYNEAWRFSNPALLMPPPGPPLPLLPLP MTATSYRGDHIKLQADSFGLHIVPV”(SEQ ID NO: 49) STS 1907 . . . 2113 /gene = “ PAX2” /standard_name = “RH80285” /db_xref = “ UniSTS:88437” STS 3105 . . . 3257 /gene = “ PAX2”/standard_name = “ D10S2478” /db_xref = “ UniSTS:74159” polyA_signal4211 . . . 4216 /gene = “ PAX2” polyA_signal 4215 . . . 4220 /gene = “PAX2” polyA_signal 4222 . . . 4227 /gene = “ PAX2” polyA_site 4240 /gene= “ PAX2”

-   ORIGIN

(SEQ ID NO: 50)    1 aggctccagt ctccggccga gtcttctcgc agccgcaacccacctggggc cagcccagag   61 ctgccagcgc cgctcggctc cctccctccc tcccggcccttcggccgcgg cggcgtgcgc  121 ctgccttttc cgggggcggg ggcctggccc gcgcgctcccctcccgcagg cgccacctcg  181 gacatccccg ggattgctac ttctctgcca acttcgccaactcgccagca cttggagagg  241 cccggctccc ctcccggcgc cctctgaccg cccccgccccgcgcgctctc cgaccaccgc  301 ctctcggatg accaggttcc aggggagctg agcgagtcgcctcccccgcc cagcttcagc  361 cctggctgca gctgcagcgc gagccatgcg cccccagtgcaccccggccc ggcccaccgc  421 cccggggcca ttctgctgac cgcccagccc cgagccccgacagtggcaag ttgcggctac  481 tgcagttgca agctccggcc aacccggagg agccccagcggggagcgcag tgttgcgccc  541 cccgcccccg cgcgccccgc agcagccggg cgttcactcatcctccctcc cccaccgtcc  601 ctcccttttc tcctcaagtc ctgaagttga gtttgagaggcgacacggcg gcggcggccg  661 cgctgctccc gctcctctgc ctccccatgg atatgcactgcaaagcagac cccttctccg  721 cgatgcaccc agggcacggg ggtgtgaacc agctcgggggggtgtttgtg aacggccggc  781 ccctacccga cgtggtgagg cagcgcatcg tggagctggcccaccagggt gtgcggccct  841 gtgacatctc ccggcagctg cgggtcagcc acggctgtgtcagcaaaatc ctgggcaggt  901 actacgagac cggcagcatc aagccgggtg tgatcggtggctccaagccc aaagtggcga  961 cgcccaaagt ggtggacaag attgctgaat acaaacgacagaacccgact atgttcgcct 1021 gggagattcg agaccggctc ctggccgagg gcatctgtgacaatgacaca gtgcccagcg 1081 tctcttccat caacagaatc atccggacca aagttcagcagcctttccac ccaacgccgg 1141 atggggctgg gacaggagtg accgcccctg gccacaccattgttcccagc acggcctccc 1201 ctcctgtttc cagcgcctcc aatgacccag tgggatcctactccatcaat gggatcctgg 1261 ggattcctcg ctccaatggt gagaagagga aacgtgatgaagttgaggta tacactgatc 1321 ctgcccacat tagaggaggt ggaggtttgc atctggtctggactttaaga gatgtgtctg 1381 agggctcagt ccccaatgga gattcccaga gtggtgtggacagtttgcgg aagcacttgc 1441 gagctgacac cttcacccag cagcagctgg aagctttggatcgggtcttt gagcgtcctt 1501 cctaccctga cgtcttccag gcatcagagc acatcaaatcagaacagggg aacgagtact 1561 ccctcccagc cctgacccct gggcttgatg aagtcaagtcgagtctatct gcatccacca 1621 accctgagct gggcagcaac gtgtcaggca cacagacatacccagttgtg actggtcgtg 1681 acatggcgag caccactctg cctggttacc cccctcacgtgccccccact ggccagggaa 1741 gctaccccac ctccaccctg gcaggaatgg tgcctgggagcgagttctcc ggcaacccgt 1801 acagccaccc ccagtacacg gcctacaacg aggcttggagattcagcaac cccgccttac 1861 taatgccgcc ccccggtccg cccctgccgc tgctgccgctgcctatgacc gccactagtt 1921 accgcgggga ccacatcaag cttcaggccg acagcttcggcctccacatc gtccccgtct 1981 gaccccaccc cggagggagg gaggaccgac gcgacgcgatgcctcccggc caccgcccca 2041 gcctcacccc atcccacgac ccccgcaacc cttcacatcacccccctcga aggtcggaca 2101 ggacgggtgg agccgtgggc gggaccctca ggcccgggcccgccgccccc agccccgcct 2161 gccgcccctc cccgcctgcc tggactgcgc ggcgccgtgagggggattcg gcccagctcg 2221 tcccggcctc caccaagcca gccccgaagc ccgccagccaccctgccgga ctcgggcgcg 2281 acctgctggc gcgcgccgga tgtttctgtg acacacaatcagcgcggacc gcagcgcggc 2341 ccagccccgg gcacccgcct cggacgctcg ggcgccaggaggcttcgctg gaggggctgg 2401 gccaaggaga ttaagaagaa aacgactttc tgcaggaggaagagcccgct gccgaatccc 2461 tgggaaaaat tcttttcccc cagtgccagc cggactgccctcgccttccg ggtgtgccct 2521 gtcccagaag atggaatggg ggtgtggggg tccggctctaggaacgggct ttgggggcgt 2581 caggtctttc caaggttggg acccaaggat cggggggcccagcagcccgc accgatcgag 2641 ccggactctc ggctcttcac tgctcctcct ggcctgcctagttccccagg gcccggcacc 2701 tcctgctgcg agacccggct ctcagccctg ccttgcccctacctcagcgt ctcttccacc 2761 tgctggcctc ccagtttccc ctcctgccag tccttcgcctgtcccttgac gccctgcatc 2821 ctcctccctg actcgcagcc ccatcggacg ctctcccgggaccgccgcag gaccagtttc 2881 catagactgc ggactggggt cttcctccag cagttacttgatgccccctc ccccgacaca 2941 gactctcaat ctgccggtgg taagaaccgg ttctgagctggcgtctgagc tgctgcgggg 3001 tggaagtggg gggctgccca ctccactcct cccatcccctcccagcctcc tcctccggca 3061 ggaactgaac agaaccacaa aaagtctaca tttatttaatatgatggtct ttgcaaaaag 3121 gaacaaaaca acacaaaagc ccaccaggct gctgctttgtggaaagacgg tgtgtgtcgt 3181 gtgaaggcga aacccggtgt acataacccc tccccctccgccccgccccg cccggccccg 3241 tagagtccct gtcgcccgcc ggccctgcct gtagatacgccccgctgtct gtgctgtgag 3301 agtcgccgct cgctgggggg gaaggggggg acacagctacacgcccatta aagcacagca 3361 cgtcctgggg gaggggggca ttttttatgt tacaaaaaaaaattacgaaa gaaaagaaat 3421 ctctatgcaa aatgacgaac atggtcctgt ggactcctctggcctgtttt gttggctctt 3481 tctctgtaat tccgtgtttt cgctttttcc tccctgcccctctctccctc tgcccctctc 3541 tcctctccgc ttctctcccc ctctgtctct gtctctctccgtctctgtcg ctcttgtctg 3601 tctgtctctg ctctttcctc ggcctctctc cccagacctggcccggccgc cctgtctccg 3661 caggctagat ccgaggtggc agctccagcc cccgggctcgccccctcgcg ggcgtgcccc 3721 gcgcgccccg ggcggccgaa ggccgggccg ccccgtcccgccccgtagtt gctctttcgg 3781 tagtggcgat gcgccctgca tgtctcctca cccgtggatcgtgacgactc gaaataacag 3841 aaacaaagtc aataaagtga aaataaataa aaatccttgaacaaatccga aaaggcttgg 3901 agtcctcgcc cagatctctc tcccctgcga gccctttttatttgagaagg aaaaagagaa 3961 aagagaatcg tttaagggaa cccggcgccc agccaggctccagtggcccg aacggggcgg 4021 cgagggcggc gagggcgccg aggtccggcc catcccagtcctgtggggct ggccgggcag 4081 agaccccgga cccaggccca ggcctaacct gctaaatgtccccggacggt tctggtctcc 4141 tcggccactt tcagtgcgtc ggttcgtttt gattctttttcttttgtgca cataagaaat 4201 aaataataat aataaataaa gaataaaatt ttgtatgtcaaaaaaaaaaa aaaaaaa

TABLE 3 Sequences of QRT-PCR Primers. Sense (5′-3′) Antisense (5′-3′)β-actin 5′-CCTGGCACCCAGCACAAT-3′ 5′-GCC GATCCACACGGAGTACT-3′ (SEQ ID NO:51) (SEQ ID NO: 52) DEFB1 5′-GTTGCCTGCCAGTC GCCAT GAGAACTTCCTAC-3′5′-TGGCCTTCCCTCTGTA ACAGGTGCCTTGAATT-3′ (SEQ ID NO: 53) (SEQ ID NO: 54)

TABLE 4 PAX2 siRNA Sequences. A pool of four siRNA was utilized toinhibit PAX2 protein expression. Sense (5′-3′) Antisense (5′-3′)Sequence A 5′-GAAGUCAAGUCGAGUCUAUUU-3′ 5′-AUAGACUCGACUUGACUUCUU-3′ (SEQID NO: 7) (SEQ ID NO: 3) Sequence B 5′-GAGGAAACGUGAUGAAGAUUU-3′5′-AUCUUCAUCACGUUUCCUCUU-3′ (SEQ ID NO: 8) (SEQ ID NO: 4) Sequence C5′-GGACAAGAUUGCUGAAUACUU-3′ 5′-GUAUUCAGCAAUCUUGUCCUU-3′ (SEQ ID NO: 9)(SEQ ID NO: 5) Sequence D 5′-CAUCAGAGCACAUCAAAUCUU-3′5′-GAUUUGAUGUGCUCUGAUGUU-3′ (SEQ ID NO: 10) (SEQ ID NO: 6)

TABLE 5 Quantitative RT-PCR Primers. Nucleotide sequences of primersused to amplify PAX2 and GAPDH. Sense (5′-3′) Antisense (5′-3′) GAPDH5′-CCACCCATGGCAAATTCCATGGCA-3′ 5′-TCTAGACGGCAGGTCAGGTCAACC-3′ (SEQ IDNO: 55) (SEQ ID NO: 56) BAD 5′-CTCAGGCCTATGCAAAAAGAGGA-3′5′-GCCCTCCCTCCAAAGGAGAC-3′ (SEQ ID NO: 57) (SEQ ID NO: 58) BID5′-AACCTACGCACCTACGTGAGGAG-3′ 5′-CGTTCAGTCCATCCCATTTCTG-3′ (SEQ ID NO:59) (SEQ ID NO: 60) BAX 5′-GACACCTGAGCTGACCTTGG-3′5′-GAGGAAGTCCAGTGTCCAGC-3′ (SEQ ID NO: 61) (SEQ ID NO: 62)

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

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It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1-11. (canceled)
 12. A method for treating cancer in a subject,comprising: administering to said subject an effective amount of ananti-PAX2 agent, wherein said anti-PAX2 agent inhibits PAX2 expressionor the binding of PAX2 to DEFB1 promoter.
 13. The method of claim 12,wherein said anti-PAX2 agent comprises a PAX2-specific siRNA.
 14. Themethod of claim 13, wherein said PAX2-specific siRNA is selected fromthe group consisting of: AUAGACUCGACUUGACUUCUU (SEQ ID NO: 3)AUCUUCAUCACGUUUCCUCUU (SEQ ID NO: 4) GUAUUCAGCAAUCUUGUCCUU (SEQ ID NO:5) GAUUUGAUGUGCUCUGAUGUU. (SEQ ID NO: 6)


15. The method of claim 12, wherein said anti-PAX2 agent comprises aviral vector capable of expressing a PAX2-specific siRNA.
 16. The methodof claim 12, wherein said anti-PAX2 agent comprises a plasmid vectorcapable of expressing a PAX2-specific siRNA.
 17. The method of claim 12,wherein said anti-PAX2 agent comprises an oligonucleotide containing aPAX2 binding site of DEFB1 promoter, wherein said PAX2 binding site ofDEFB1 promoter comprises the nucleotide sequence of SEQ ID NO:1 or thenucleotide sequence complementary to SEQ ID NO:1.
 18. The method ofclaim 17, wherein said anti-PAX2 agent further comprise a liposome. 19.The method of claim 17, wherein said PAX2 binding site of DEFB1 promotercomprises a contiguous DEFB1 promoter sequence of 6-75 nucleotides. 20.The method of claim 19, wherein said contiguous DEFB1 promoter sequenceis selected from the group consisting of SEQ ID NOS: 16, 18-21 andnucleotide sequence complementary to SEQ ID NOS: 16 and 18-21.
 21. Themethod of claim 12, wherein said anti-PAX2 agent is administered with apharmaceutically acceptable carrier.
 22. The method of claim 21, whereinsaid anti-PAX2 agent is administered parenterally.
 23. The method ofclaim 12, wherein said anti-PAX2 agent is conjugated to an antibody, areceptor or a receptor ligand to target tumor tissue in said subject.24. The method of claim 12, wherein said anti-PAX2 agent comprises ananti-PAX2 antibody.
 25. The method of claim 12, wherein said anti-PAX2agent is a small molecule that interferes or inhibit binding of PAX2 tothe DEFB1 promoter.
 26. The method of claim 12, wherein said cancer isprostate cancer.
 27. A method for treating cancer in a subject,comprising: administering an effective amount of DEFB1 enhancing agentto said subject.
 28. The method of claim 27, wherein said DEFB1enhancing agent comprises DEFB1 protein.
 29. The method of claim 27,wherein said DEFB1 enhancing agent comprises a DEFB1-encoding nucleicacid capable of expressing DEFB1 in a cell.
 30. The method of claim 27,wherein said of DEFB1 enhancing agent comprises a PAX2-inhibitor. 31.The method of claim 30, wherein said PAX2-inhibitor inhibits PAX2expression.
 32. The method of claim 31, wherein said PAX2-inhibitor is aPAX2-specific siRNA.
 33. The method of claim 30, wherein saidPAX2-inhibitor is an oligonucleotide that inhibits the binding of PAX2to DEFB1 promoter.
 34. The method of claim 33, wherein saidPAX2-inhibitor is an oligonucleotide comprising the core PAX2 bindingsequence of DEFB1 promoter.
 35. A composition for treating cancer,comprising a PAX2-specific siRNA, an agent capable of expressing aPAX2-specific siRNA in vivo, wherein said siRNA is selected from thegroup consisting of: AUAGACUCGACUUGACUUCUU (SEQ ID NO: 3)AUCUUCAUCACGUUUCCUCUU (SEQ ID NO: 4) GUAUUCAGCAAUCUUGUCCUU (SEQ ID NO:5) GAUUUGAUGUGCUCUGAUGUU. (SEQ ID NO: 6)


36. The composition of claim 35, wherein said agent is a viral vectorcarrying a coding sequence of said siRNA operably linked to a regulatoryelement.
 37. The composition of claim 35, wherein said agent is aplasmid containing a coding sequence of said siRNA operably linked to aregulatory element.
 38. The composition of claim 37, further comprisinga non-viral carrier.
 39. The composition of claim 38, wherein saidnon-viral carrier is a liposome.
 40. The composition of claim 35,further comprising a pharmaceutically acceptable carrier.
 41. Acomposition for treating cancer, comprising a DEFB1 enhancing agent anda pharmaceutically acceptable carrier.
 42. The composition of claim 41,wherein said DEFB1 enhancing agent comprises DEFB1 protein.
 43. Thecomposition of claim 41, wherein said DEFB1 enhancing agent comprises aDEFB1-encoding nucleic acid capable of expressing DEFB1 in a cell. 44.The composition of claim 41, wherein said DEFB1 enhancing agentcomprises a PAX2-inhibitor.
 45. The composition of claim 44, whereinsaid PAX2-inhibitor inhibits PAX2 expression.
 46. The composition ofclaim 44, wherein said of PAX2-inhibitor is a PAX2-specific siRNA. 47.The composition of claim 44, wherein said of PAX2-inhibitor is anoligonucleotide that inhibits the binding of PAX2 to DEFB1 promoter.